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Tuberculosis Teaching Basics
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DrTVRao MD 1
HISTORY ofTuberculosis
Tuberculosis Is an Ancient Disease
Spinal Tuberculosis in Egyptian
Mummies History dates to
1550 ndash 1080 BC Identified by
PCR
DrTVRao MD 2
Robert Koch Discoverer of
MycobacteriumTuberculosis
DrTVRao MD 3
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 4
Classification of Mycobacteria1 Tubercle bacilli
a) Human ndash MTBb) Bovine ndash M bovisc) Murine ndash M microtid) Avian ndash M aviume) Cold blooded ndash M
marinum2 Lepra bacilli
a) Human ndash M lepraeb) Rat ndash M leprae murium
3 Mycobacteria causing skin ulcers
a) M ulceransb) M belnei
4 Atypical Mycobacteria (Runyon Groups)
a) Photochromogensb) Scotochromogensc) Nonphotochromogensd) Rapid growers
5 Johnersquos bacillusM paratuberculosis
6 Saprophytic mycobacteriaa) M butyricumb) M phleic) M stercoralisd) M smegmatise) Others
DrTVRao MD 5
Mycobacterium differ from other routinely isolated Bacteria
bull Slow-growing with a generation time of 12 to 18 hours (cf 20-30 minutes for Escherichia coli)
bull Hydrophobic with a high lipid content in the cell wall Because the cells are hydrophobic and tend to clump together they are impermeable to the usual stains eg Grams stain
DrTVRao MD 6
Acid fast bacillibull Known as ldquoAcid-fast bacilli
because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 7
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
HISTORY ofTuberculosis
Tuberculosis Is an Ancient Disease
Spinal Tuberculosis in Egyptian
Mummies History dates to
1550 ndash 1080 BC Identified by
PCR
DrTVRao MD 2
Robert Koch Discoverer of
MycobacteriumTuberculosis
DrTVRao MD 3
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 4
Classification of Mycobacteria1 Tubercle bacilli
a) Human ndash MTBb) Bovine ndash M bovisc) Murine ndash M microtid) Avian ndash M aviume) Cold blooded ndash M
marinum2 Lepra bacilli
a) Human ndash M lepraeb) Rat ndash M leprae murium
3 Mycobacteria causing skin ulcers
a) M ulceransb) M belnei
4 Atypical Mycobacteria (Runyon Groups)
a) Photochromogensb) Scotochromogensc) Nonphotochromogensd) Rapid growers
5 Johnersquos bacillusM paratuberculosis
6 Saprophytic mycobacteriaa) M butyricumb) M phleic) M stercoralisd) M smegmatise) Others
DrTVRao MD 5
Mycobacterium differ from other routinely isolated Bacteria
bull Slow-growing with a generation time of 12 to 18 hours (cf 20-30 minutes for Escherichia coli)
bull Hydrophobic with a high lipid content in the cell wall Because the cells are hydrophobic and tend to clump together they are impermeable to the usual stains eg Grams stain
DrTVRao MD 6
Acid fast bacillibull Known as ldquoAcid-fast bacilli
because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 7
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Robert Koch Discoverer of
MycobacteriumTuberculosis
DrTVRao MD 3
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 4
Classification of Mycobacteria1 Tubercle bacilli
a) Human ndash MTBb) Bovine ndash M bovisc) Murine ndash M microtid) Avian ndash M aviume) Cold blooded ndash M
marinum2 Lepra bacilli
a) Human ndash M lepraeb) Rat ndash M leprae murium
3 Mycobacteria causing skin ulcers
a) M ulceransb) M belnei
4 Atypical Mycobacteria (Runyon Groups)
a) Photochromogensb) Scotochromogensc) Nonphotochromogensd) Rapid growers
5 Johnersquos bacillusM paratuberculosis
6 Saprophytic mycobacteriaa) M butyricumb) M phleic) M stercoralisd) M smegmatise) Others
DrTVRao MD 5
Mycobacterium differ from other routinely isolated Bacteria
bull Slow-growing with a generation time of 12 to 18 hours (cf 20-30 minutes for Escherichia coli)
bull Hydrophobic with a high lipid content in the cell wall Because the cells are hydrophobic and tend to clump together they are impermeable to the usual stains eg Grams stain
DrTVRao MD 6
Acid fast bacillibull Known as ldquoAcid-fast bacilli
because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 7
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 4
Classification of Mycobacteria1 Tubercle bacilli
a) Human ndash MTBb) Bovine ndash M bovisc) Murine ndash M microtid) Avian ndash M aviume) Cold blooded ndash M
marinum2 Lepra bacilli
a) Human ndash M lepraeb) Rat ndash M leprae murium
3 Mycobacteria causing skin ulcers
a) M ulceransb) M belnei
4 Atypical Mycobacteria (Runyon Groups)
a) Photochromogensb) Scotochromogensc) Nonphotochromogensd) Rapid growers
5 Johnersquos bacillusM paratuberculosis
6 Saprophytic mycobacteriaa) M butyricumb) M phleic) M stercoralisd) M smegmatise) Others
DrTVRao MD 5
Mycobacterium differ from other routinely isolated Bacteria
bull Slow-growing with a generation time of 12 to 18 hours (cf 20-30 minutes for Escherichia coli)
bull Hydrophobic with a high lipid content in the cell wall Because the cells are hydrophobic and tend to clump together they are impermeable to the usual stains eg Grams stain
DrTVRao MD 6
Acid fast bacillibull Known as ldquoAcid-fast bacilli
because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 7
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Classification of Mycobacteria1 Tubercle bacilli
a) Human ndash MTBb) Bovine ndash M bovisc) Murine ndash M microtid) Avian ndash M aviume) Cold blooded ndash M
marinum2 Lepra bacilli
a) Human ndash M lepraeb) Rat ndash M leprae murium
3 Mycobacteria causing skin ulcers
a) M ulceransb) M belnei
4 Atypical Mycobacteria (Runyon Groups)
a) Photochromogensb) Scotochromogensc) Nonphotochromogensd) Rapid growers
5 Johnersquos bacillusM paratuberculosis
6 Saprophytic mycobacteriaa) M butyricumb) M phleic) M stercoralisd) M smegmatise) Others
DrTVRao MD 5
Mycobacterium differ from other routinely isolated Bacteria
bull Slow-growing with a generation time of 12 to 18 hours (cf 20-30 minutes for Escherichia coli)
bull Hydrophobic with a high lipid content in the cell wall Because the cells are hydrophobic and tend to clump together they are impermeable to the usual stains eg Grams stain
DrTVRao MD 6
Acid fast bacillibull Known as ldquoAcid-fast bacilli
because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 7
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Mycobacterium differ from other routinely isolated Bacteria
bull Slow-growing with a generation time of 12 to 18 hours (cf 20-30 minutes for Escherichia coli)
bull Hydrophobic with a high lipid content in the cell wall Because the cells are hydrophobic and tend to clump together they are impermeable to the usual stains eg Grams stain
DrTVRao MD 6
Acid fast bacillibull Known as ldquoAcid-fast bacilli
because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 7
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Acid fast bacillibull Known as ldquoAcid-fast bacilli
because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 7
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
How they are Acid fastbull Once stained the cells resist
decolourization with acidified organic solvents and are therefore called acid-fast (Other bacteria which also contain mycolic acids such as Nocardia can also exhibit this feature)
DrTVRao MD 8
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Mycobacterium tuberculosis complex
bull Includes Human and Bovine mycobacterium
bull M Africanism Tropical Africabull Mmicroti do not cause human
infections but in small mammals
DrTVRao MD 9
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Mbovisbull Primarily infection among the
cattlebull Mbovis infects Tonsils Cervical
nodes can produce Scrofulabull Enter through Intestines ndash infects
the Ileocecal regionDrTVRao MD 10
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
What are atypical MycobacteriumWhat are atypical Mycobacterium
bull Infects birds cold blooded animals worm blooded animals
bull Present in environmentbull Opportunistic pathogensbull Others ndash Saprophytic bacteria M butryicum present in butter Mphlei M smegmatis ndash present in Smegma
DrTVRao MD 11
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Atypical Mycobacterium
bull 1 Photochromogensbull 2 Scotochromogensbull 3 Non Photochromogensbull 4 Rapid growers
DrTVRao MD 12
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
MOST IMPORTANT AMONG INFECTIOUS DISEASES
bull Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease The disease affects 18 billion peopleyear which is equal to one-third of the entire world population
DrTVRao MD 13
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Poverty and Crowded living spreads Tuberculosis
DrTVRao MD 14
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculosis infects Famous people too
DrTVRao MD 15
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
What are Mycobacteria
bull Obligate aerobes growing most successfully in tissues with a high oxygen content such as the lungs
bull Facultative intracellular pathogens usually infecting mononuclear phagocytes (eg macrophages)
DrTVRao MD 16
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
General characters of the genus
bull Slender rodsbull Resist staining but
once stained resist decolonization by dilute mineral acids hence called ACID FAST BACILLI (AFB)
bull Aerobic Non-motile Non-sporing Non-capsulated
bull Growth generally slowbull Genus includes
ndash Obligate parasitesndash Opportunist pathogensndash Saprophytes
DrTVRao MD 17
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Morphology of Mycobacterium tuberculosis
bull Straight slightly curved Rod shaped 3 x 03microns
bull May be single in pairs or in small clumps
bull On conditions in growth appears as filamentous club shaped or in Branched forms
DrTVRao MD 18
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
ACID FAST BACILLI
bull Known as ldquoAcid-fast bacilli because of their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol
DrTVRao MD 19
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Important MycobacteriumImportant Mycobacterium
bull Mycobacterium tuberculosis along with M bovis M africanum and M microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex Each member of the TB complex is pathogenic but M tuberculosis is pathogenic for humans while M bovis is usually pathogenic for animals
DrTVRao MD 20
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Avian Tuberculosis
bull Transmitted by ingestion and inhalation of aerosolized infectious organisms from feces
bull Oral ingestion of food and water contaminated with feces is the most common method of infection
bull Once ingested the organism spreads throughout the birds body and is shed in large numbers in the feces
bull If the bacterium is inhaled pulmonary lesions and skin invasions may occur
bull transmission of avian TB is from bird to human not from human to human
DrTVRao MD 21
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Acid Fast Bacilli seen in a specimen of Sputum
DrTVRao MD 22
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Acid fast bacilli
DrTVRao MD 23
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Acid fast Bacilli seen as in Florescent Microscope
bull After staining with Ziehl Neelsen method or Fluorescent method ( Auramine or Rhodamine they resist decolonization by 20 Sulphuric acid and absolute alcohol for 10 mt
bull So called as Acid and Alchool fast
DrTVRao MD 24
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Why they are Acid Fast
bull The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell)
DrTVRao MD 25
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
MTB Cultural charactersbull Grow slowly
Generation time 14-15 hrs
bull Colonies appear after 2 weeks or at 6-8 weeks
bull MTB - Obligate aerobe
bull MTB grows more luxuriantly (eugonic) than M bovis (dysgonic)
bull Addition of 05 Glycerol supports growth of human strains No effect or inhibitory effect on bovine strains
DrTVRao MD 26
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Culturing Acid Fast Bacillibull Slow to grow bull Generation time is 14 ndash
15 hoursbull gt 2 weeks minimal
required periodbull Grows at 370c do not
grow below 250cbull Ph between 64 to 70
DrTVRao MD 27
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
DrTVRao MD 28
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Nature of Media Used
bull Helps the growth needsbull Solid Medium is commonly usedbull Lowenstein Jensenrsquos mediumbull Petranginibull Middle brook medium
DrTVRao MD 29
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Lowenstein Jensenrsquos Medium
bull Contain coagulated egg
bull Mineral salt solution
bull Asparaginesbull Malachite greenbull Agar
DrTVRao MD 30
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Other Medium
bullMiddle brookbullSulas mediumbullBut not routinely used
DrTVRao MD 31
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Nature of Growth CharactersNature of Growth Charactersbull M tuberculosis is obligate aerobebull Mbovis Microaerophilicbull Mtuberculosis growth luxierentlybull Mtuberculosis eugonicbull M bovis is dysgonicbull When grown on 05 glycerin M tuberculosis growth
improvesbull Sodium pyruvate improves the growth of both
organism
DrTVRao MD 32
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
On L J Mediumbull Mtuberculosis appear
dry rough raised irregular colonies
bull Appear wrinkledbull They appear creamy
whitebull Become yellowishbull Mbovis appear as flat
smooth moist white and break up easily
DrTVRao MD 33
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Lowenstein Jensen Medium ndash
Selective Always in screw capped bottle Bluish Green
Contains ndash Egg protein ndash Solidifying agent
Mineral salts ndash Mg Sulphate Mg citrate
Asparagine
Malachite Green ndash Selective agent
Sterilized by - Inspissation
DrTVRao MD 34
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
On Liquid MediumbullAppear as long serpentine cords in liquid medium
bullVirulent strains grow in a more dispersed manner
DrTVRao MD 35
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Resistance of Mycobacteriumbull Mycobacterium are killed at 600c in 15 ndash 20 mtbull In sputum they survive for 10 ndash 30 mtbull Relatively resistant to several chemicals
including Phenol 5 bull Sensitive to Glutaraldehyde and
Formaldehydebull Ethanol is suitable application to superficial
surfaces and skin gloves
DrTVRao MD 36
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Resistance to several agents
bull Bacilli survive in Droplets for 8 ndash 10 daysbull Survive in 5 phenol 15 Sulphuric acid 3 Nitric acid5 oxalic acid 4 Sodium hydroxide
DrTVRao MD 37
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Biochemical Tests on Mycobacterium spp
bull Niacin test ndash 10 cyanogens bromide and 4 Aniline in 96 ethanol are added to suspension of ndash C canary yellow color indicates positive test
DrTVRao MD 38
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Other Testsbull Aryl sulphatase test ndash Positive in Atypical
Mycobacterium bull Bacilli grown in 0001 tripotassium phenolpthalein
disulphide 2 N Sodium hydroxide added drop by drop a pink color develops
bull Catalase peroxidase test ndash Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive ndash not atypical INH resistant strains are negative for test
DrTVRao MD 39
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Catalase Testbull 30 Vol of H2O2 and 02 alcohol in
distilled water is added to 5 ml of test culture
bull Effervescence indicates Catalase positivebull Other test Amidase test Nitrate reduction test
DrTVRao MD 40
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Antigenic Charactersbull Group specificity due to Polysaccharidesbull Type specificity to protein antigensbull Delayed hypersensitivity to proteinsbull Related to each other species bull Some relation between lepra and tubercle bacillibull Serology ndash Tests not useful Antigenic homogeneity between lt bovis and
Mmicroti
DrTVRao MD 41
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Bacteriophagesbull There are 4 Bacteriophages A B C Dbull A worldwidebull B Europe and -Americanbull C rarebull I type nature between A and B and common in
Indiabull Phage 33 D M tuberculosis and not in BCG
strainsDrTVRao MD 42
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Molecular Typingbull DNA finger printing
differentiates different strains of Mycobacterium species
bull Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific
bull Use in epidemiological studies
DrTVRao MD 43
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Finger printing Methods
bull Finger printing is done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli
bull Now entire genome of M tuberculosis is sequenced
bull Several Molecular methods are available for studies
DrTVRao MD 44
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Genome of Mycobacterium tuberculosis
DrTVRao MD 45
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
How tuberculosis
spreadsbull Tuberculosis (TB) is a contagious disease Like the common cold it spreads through the air Only people who are sick with TB in their lungs are infectious When infectious people cough sneeze talk or spit they propel TB germs known as bacilli into the air A person needs only to inhale a small number of these to be infected
DrTVRao MD 46
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculosis spread by Respiratory route
DrTVRao MD 47
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculosis highly Communicable Disease
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 48
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
In India 1 death Minute bull Half a million
people die from disease every year in India one death every minute
DrTVRao MD 49
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Pathology and Pathogenesis ofTuberculosis
bull Source of Infection ndash Open case of Pulmonary Tuberculosis
bull Every open case has potential to infect 20 ndash 25 healthy persons before cured or dies
bull Coughing Sneezing or Talkingbull Each act can spill 3000 infective nuclei in the
airbull Infective particles are engulfed by Alveolar
Macrophages
DrTVRao MD 50
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Spread of Tuberculosis
DrTVRao MD 51
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Generation of Droplet Nucleibull One cough produces
500 dropletsbull The average TB patient
generates 75000 droplets per day before therapy
bull This falls to 25 infectious droplets per day within two weeks of effective therapy
DrTVRao MD 52
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
DrTVRao MD 53
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Predisposing Factors
bull Genetic basisbull Age bull Stressbull Nutritionbull Co existing infections Eg HIV
DrTVRao MD 54
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Mechanisms of Infection
bull Mycobacterium do not produce toxinsbull Allergy and Immunity plays the major rolebull Only 110 of the infected will get diseasebull Cell Mediated Immunity plays a crucial rolebull Humoral Immunity ndash not Importantbull CD4 Cell plays role in Immune Mechanisms
DrTVRao MD 55
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Mechanisms of Infectionbull Within 10 days of entry of Bacilli
clones of Antigen specific T Lymphocytes are produced
bull Can actively produce Cytokines Interferon γ which activate
Macrophages form cluster or Granuloma
DrTVRao MD 56
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activatedmacrophage
Lymphocyte
Fully activated macrophage
DrTVRao MD 57
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Basis of Tubercle formationbull Tubercle is a Avascular granuloma
Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts
bull Produce lesions may be Exudative or Productive
DrTVRao MD 58
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Diagram of a
Granuloma
NOTE ultimately a fibrin layer
develops around granuloma
(fibrosis) further ldquowalling offrdquo the
lesion
Typical progression in pulmonary
TB involves caseation
calcification and cavity
formation
DrTVRao MD 59
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tubercle discharging
Bronchial tree
TNF- TNF- DrTVRao MD 60
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Immunity in Tuberculosisbull CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines2 Interleukin 1and 2 3 Interferons γ 4Tumor necrosis factor
bull The mechanisms with Th 1 secrete Cytokines Activate Macrophages Results in protective Immunity and contain Infection Th 2 manifests with Delayed Hypersensitivity DTH causes Tissue destruction and disease will
progress
DrTVRao MD 61
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Immunity in Tuberculosis Activated Macrophages - Epitheliod cellsForms cluster a granuloma Activated macrophages turn into Giant cellsGranuloma contains necrotic tissue Dead macrophages cheese like caseationApoptosis of bacteria laden cellsContribute to protective immunity
DrTVRao MD 62
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Lesions in Tuberculosisbull Exudative ndash and Productive
bull Exudative ndash Acute inflammatory reaction with edema fluid ndash contains Polymorphs-
Lymphocytes ndash later Mononuclear cellsBacilli are virulent - Host responds with DTH
Injurious
Productive Type protective Immunity
DrTVRao MD 63
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Primary Tuberculosisbull Initial response bull In Endemic countries Young children bull Events of Primary complex 1 Bacilli are engulfed by Alveolar Macrophages 2 Multiply and give raise to Sub pleural focus of
TuberculosisPneumoniainvolve lower lobes and lower part of upper lobes
Called as Ghonrsquos focusThe Hilar Lymph nodes are also involved
DrTVRao MD 64
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Primary complex
bull This is known as the primary complex or primary infection The patient will heal and a scar will appear in the infected loci There will also be a few viable bacilli may remain in these areas (particularly in the lung) The bacteria at this time goes into a dormant state as long as the persons immune system remains active and functions normally this person isnt bothered by the dormant bacillus
DrTVRao MD 65
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Primary complexbull Ghonrsquos focus with Enlarged lymph nodes appear
after 3- 8 weeks after infectionbull Heals in 2 ndash 6 months calcifiedbull Some bacteria remain alive and produce latent
infectionsbull Infection activated in Immunosuppressed conditions
Eg HIV infections and AIDSbull Can produce Meningitis Miliary tuberculosis other
disseminated Tuberculosis
DrTVRao MD 66
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Reactivation of Tuberculosisbull When a persons
immune system is depressed a secondary reactivation occurs 85-90 of the cases seen which are of secondary reactivation type occurs in the lungs
DrTVRao MD 67
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Kochrsquos Phenomenonbull Tuberculosis infected Guinea pig if injected
with Living Tubercle bacillibull The site around the injection becomes
necroticbull Koch found the same reaction when injected
with old Tuberculin ( heated and concentration of the tubercle bacilli )
bull It has produced the same reactionbull This is called as Kochrsquos Phenomenon
DrTVRao MD 68
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Post Primary Tuberculosisbull Mainly occurs due to Reactivation of Latent
infectionbull May also due to Exogenous reinfectionbull Differs from Primary Infectionbull Leads to ndash Cavitations of Lungs Enlargement of Lymph
nodesExpectoration of Bacteria laden sputumDissemination into Lungs and other extra pulmonary
areas
DrTVRao MD 69
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Majority of the Tuberculosisare Pulmonary
DrTVRao MD 70
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Multiorgan Involvementin Tuberculosis
DrTVRao MD 71
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Complication of Tuberculosis
1 Meningitis2 Pleurisy3 Involvement of Kidney4 Spine ( Potts spine )5 Bone Joints6 Miliary tuberculosis
DrTVRao MD 72
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Symptoms and Sings of Tuberculosis
DrTVRao MD 73
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Clinical Illness with Tuberculosis
bull Pulmonary Disease ndash Major manifestation with involvement of Lungs
Hemoptysis Chest pain Fever sweets
Anorexia Cavity formation in
Lungs
DrTVRao MD 74
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculosis - Pneumothorax
DrTVRao MD 75
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Extra pulmonary Tuberculosis
bull Bacteria on circulation leads to bacteremia leads to involvement of
GUT Genito urinary system Meningitis Gastro Intestinal system skin Lymph
nodes Bone marrow Spinal infection Potts spine Arthritis
DrTVRao MD 76
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculosis - Lymphadenitis
DrTVRao MD 77
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Multidrug-resistant tuberculosis (MDR-TB)
bull Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients This improper use is a result of a number of actions including administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment
DrTVRao MD 78
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Definition of MDR Tuberculosis
bull MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first-line drugs (FLD) XDR-TB is defined as resistance to at least isoniazid and rifampicin and to any fluoroquinolone and to any of the three second-line injectables (amikacin capreomycin and kanamycin)
DrTVRao MD 79
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
MDR tuberculosis dangerous to Society too
bull Essentially drug resistance arises in areas with weak TB control programmes A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals
DrTVRao MD 80
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
X- MDRbull The term lsquototally drug resistantrsquo has not
been clearly defined for tuberculosis XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited
DrTVRao MD 81
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Epidemiologybull An ancient disease called as white plaguebull 13 of the world population is infected bull 2 billion infectedbull Each year 9 lakhs to 1 million are infectedbull Poor nations phase the burnt of the diseasebull In developing world gt 4o of the population is
effectedbull 15 million suffer the diseasebull 3 million are highly infective
DrTVRao MD 82
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Diagnosis of Tuberculosis
DrTVRao MD 83
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Types of specimens-Sputum
- BAL
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric ndash cold abscess)
- Blood in case of haematogenous TB
DrTVRao MD 84
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
85
Sputum Collection
bull Sputum specimens are essential to confirm TBndash Specimens should be from lung secretions not
salivabull Collect 3 specimens on 3 different daysbull Spontaneous morning sputum more
desirable than induced specimensbull Collect sputum before treatment is
initiated
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
86
Culturebull Used to confirm diagnosis of TBbull Culture all specimens even if smear is
negativebull Initial drug isolate should be used to
determine drug susceptibility
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Laboratory Diagnosis1- Sputum smears stained by Z-N stain
Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB
Advantage - cheap ndash rapid - Easy to perform - High predictive value gt 90 - Specificity of 98Disadvantages - sputum ( need to contain 5000-10000 AFB ml) - Young children elderly amp HIV infected persons
may not produce cavities amp sputum containing AFBDrTVRao MD 87
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
2- Detecting AFB by fluorochrome stain using fluorescence microscopy
The smear may be stained by aura mine-O dye In this method the TB bacilli are stained yellow against dark background amp easily visualized using florescent microscope
Advantages - More sensitive - RapidDisadvantages - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain
DrTVRao MD 88
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Quantitation of AFB in Sputum Smears
bull No of Bacilli No of Fields Report asbull 0 300 Negativebull 1-2 300 Doubtfulbull 1- 9 100 1+bull 1- 9 10 2+bull 1-9 1 3+bull 10 or gt10 1 4+
DrTVRao MD 89
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
LowensteinndashJensen mediumbull When grown on LJ medium
M tuberculosis appears as brown granular colonies (sometimes called buff rough and tough) The media must be incubated for a significant length of time usually four weeks due to the slow doubling time of M tuberculosis
DrTVRao MD 90
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
3- Cultures on L J media Lowenstein ndashJensen medium is an egg based media
with addition of salts 5 glycerol Malachite green
Advantages - Specificity about 99 - More sensitive (need lower no of bacilli 10-100
ml) - Can differentiate between TB complex amp
NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St INH Rif E) Disadvantages Slowly growing ( up to 8 weeks ) DrTVRao MD 91
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Detection and identification of mycobacteria directly
from clinical samplesbull Genotypic Methods bull 1048708 PCRbull 1048708 LAMPbull 1048708 TMA NAAbull 1048708 Ligase chain reactionbull 1048708 Phenotypic Methods bull 1048708 FAST Plaque TB
DrTVRao MD 92
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
bull Essentially PCR is a way to make millions of identical copies of a specific DNA sequence which may be a gene or a part of a gene or simply a stretch of nucleotides with a known DNA sequence the function of which may be unknown
Polymerase Chain Reaction (PCR)
DrTVRao MD 93
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
bull Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole bloodPBMCs incubated with TB antigens
Quantiferon-GOLD
DrTVRao MD 94
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculin TestInterpretation A positive test indicates previous exposure and
carriage of TB A negative tuberculin test excludes infection in
suspected persons Tuberculin positive persons may develop
reactivation type of TB Tuberculin negative persons are at risk of gaining
new infection False positive reactions are mainly due to - Infection with nontuberculous mycobacteria
DrTVRao MD 95
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
False negative reactions may be due to -
bull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB)
- Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition
- AIDSbull Children below 5 years of age with no
exposure historybull - Positive test must be regarded suspicious
DrTVRao MD 96
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculin Test( Mantoux Test )
bull Test to be interpreted in relation to clinical evaluation
bull Even the induration of 5 mm to be considered positive when tested on HIV patients
bull Lacks specificity
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculin Testing - Limitations
bull False positive reactions are mainly due tobull - Infection with nontuberculous mycobacteriabull False negative reactions may be due tobull - Sever tuberculosis infection (Miliary TB) -
Hodgkinrsquos disease bull - Corticosteroid therapy - Malnutrition -
AIDSbull Children below 5 years of age with no exposure
historybull - Positive test must be regarded suspicious
DrTVRao MD 98
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Recent Methods for DiagnosisI ndash BACTEC 460 ( rapid radiometric culture system )
specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 ndash labeled palmitic acid amp PANTA antibiotic mixture
Growing mycobacteria utilize the acid releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument
The daily increase in GI output is directly proportional to the rate amp amount of growth in the medium
DrTVRao MD 99
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
III Polymerase Chain Reaction (PCR) amp Gene probe
Nucleic acid probes amp nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells
Advantages
- Rapid procedure - High sensitivity (1-10
( 3 ndash 4 hours) bacilli ml sputum)
DrTVRao MD 100
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Tuberculosis and HIV infection
bull HIV association has become a threat to the developed countries too
bull HIV association will lead to rapid spread of tuberculosis
DrTVRao MD 101
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
HIV Considerationsbull HIV is the strongest risk factor for
progression to active diseasebull HIV kills CD4+ T Helper cells which
normally inhibit M tuberculosisbull HIV interferes with PPD skin testbull Protease inhibitors interfere with
rifampin
DrTVRao MD 102
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
MDR tuberculosisbull Multidrug resistant tuberculosis has become a
global threatbull In 1993 WHO declared Tuberculosis a Global
emergencybull Animals shed the bacilli in Milk humanrsquos get
infected after drinking the unsterilized Milkbull Pasteurization has reduced the incidence of
Bovine tuberculosis
DrTVRao MD 103
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Second Line Drug Treatment (SLDrsquos)
Less effective more costly and more toxic 50 cure rate
bull Four months intensive phase (5 drugs)ndash Kanamycinndash Ethionamidendash Pyrazinamidendash Ofloxacinndash Cycloserine or Ethambutol
bull 7X times pw in hospital raquo Followed by
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Why Tuberculosis continues to be Important
bull Someone in the world is newly infected with TB bacilli every second
bull Overall one-third of the worlds population is currently infected with the TB bacillus
bull 5-10 of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life People with HIV and TB infection are much more likely to develop TB
DrTVRao MD 105
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
March 24th World TB Day
DrTVRao MD 106
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
TreatmentDrugs used 1- First line drugs - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective) - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin
Noncompliance (failure to complete the course) Directly observed therapy (DOT) Health care workers observe the medication
DrTVRao MD 107
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Directly Observed Therapy ndash Short Course
bull DOTS (directly observed treatment short-course) is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components
bull Case detection by sputum smear microscopybull Standardized treatment regimen directly
observed by a healthcare worker or community health worker for at least the first two months
bull A regular drug supplyDrTVRao MD 108
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Immuno-prophylaxis
bull Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG)
bull The strain causes self limited lesion and induces hypersensitivity amp immunity
bull Coverts tuberculin negative person to positive reactor
bull Immunity lasts for 10-15 years Immunity 60-80bull Some studies proved BCG is doubtful value in
prevention of TuberculosisDrTVRao MD 109
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
Bacillus Calmette-Gueacuterin (BCG)
bull Bacillus Calmette-Gueacuterin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus Mycobacterium bovis that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years
DrTVRao MD 110
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
BCGbull Given at birth without tuberculin testingbull Protects against TB the disease runs
milder course in protected prevents skeletal meningeal amp miliary forms
bull Also found useful in leprosy leukaemias and other malignancies by non-specific stimulation of RE system
DrTVRao MD 111
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
bull Programme Created by DrTVRao MD for Medical and Paramedical Students in
the Developing Worldbull Email
bull doctortvraogmailcom
DrTVRao MD 112
