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Shyam Kumar MishraAssistant Professor, Institute of Medicine

Laboratory diagnosis of LoWEr rEsPiratory traCt infECtions inCLuding tubErCuLosis

RESPIRATORY TRACT: ANATOMICAL STRUCTURE

It is utmost important to be familiar with the anatomic structure of the thoracic cavity, so that specimens collected from various sites in the lower respiratory tract are appropriately processed by the laboratory.

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ANATOMY OF RESPIRATORT TRACT

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INITIATION………

Aspiration of colonizing flora into the alveoli Inhalation of aerosols Hematologic seeding of the lung from a distant

focus

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When paediatric patients with cystic fibrosis (CF) have respiratory infections (exacerbations), the trigger is most commonly related to an increased growth of bacteria that are chronically present in the airway. 

However, in adult patients with COPD, the cause of respiratory exacerbations is usually related to the acquisition of new strains of bacteria, rather than an increase in the number of bacteria present when clinically stable.

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1- Bronchitis.A- Acute:

Usually caused by viruses.

In infants & preschool children is Bordetella pertussis.

B- Chronic:

It is defined by clinical symptoms in which excessive mucus production leads to coughing up sputum on most days during at least 3 consecutive months for more than 2 successive years.

2- Bronchiolitis. It is the inflammation of smaller diameter bronchiolar epithelial surfaces.

It is an acute viral LRTI that primarily occurs during the first 2 years of life.

Diseases of the Lower Respiratory Tract

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2- Pneumonia Community-Acquired Pneumonia:

patients are believed to have acquired infection outside the hospital setting.

The etiology of acute pneumonias is strongly dependent on age. More than 80% of pneumonias in infants & children are caused by viruses, whereas 10%-20% of pneumonias in adults are viral.

Hospital-Acquired Pneumonia

It is the leading cause of death among patients with nosocomial infections (as high as 50% mortality among patients in intensive care units).

Diseases of the Lower Respiratory Tract

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RESPIRATORY TRACT PATHOGENS

Definitive Respiratory Tract Pathogens Haemophilus influenzae Streptococcus pneumoniae Mycobacterium tuberculosis Mycoplasma pneumoniae Chlamydia trachomatis Chlamydophila pneumoniae Bordetella pertussis Legionella spp. Pneumocystis jiroveci Nocardia spp. Histoplasma capsulatum Coccidioides immitis Cryptococcus neoformans Blastomyces dermatitidis Viruses ( Respiratory syncytal viruse, human metapneumovirus,

adenoviruses, enteroviruses, Hantavirus, herpes simplex virus, influenza and parainfluenza viruses, Rhinoviruses, SARS etc.)

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RESPIRATORY TRACT PATHOGENS

Rare Respiratory Tract Pathogens Francisella tularensis Bacillus anthracis Yersinia pestis Burkholderia pseudomallei Coxiella burnetti Chlamydophila psittaci Brucella spp. Pasteurella multocida Klebsiella rhinoscleromatis Varicella-zooster viruse (VZV) parasites

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PATHOGENESIS

Host Factors Nasal hairs Convoluted passage Mucus lining Secretary IgA Nonspecific antibacterial substances Cilia Reflexes such as coughing, sneezing & swallowing.

Microorganism Factors Adherence Colonization Fimbriae Toxins

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LABORATORY DIAGNOSIS

Specimen collection and TransportA. Sputum1. Expectorated Food should not have been ingested for 1-2 hours before

expectoration. Mouth should be rinsed with saline or water just before

expectoration. The patient should be standing, if possible or sitting upright in bed. He or she should take deep breath to full the lungs, and empty then in one

breath, coughing as hard and as deeply as possible. Sputum brought up should be spit into screw capped container. Visually inspect the specimen. Tighten the cap of the container and send immediately to lab

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Sputum of less than 2ml should not be processed unless obviously purulent

Only 1 sputum per 24hr submitted

SPUTUM COLLECTIOND

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TRANSPORTATION OF SPUTUM

Transportation in <2 hr is recommended with refrigeration if delays anticipated.

Handle all samples using universal precautions.

Perform Gram stain and plant specimen as soon as possible

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2. Induced Assisted by respiratory therapy technician. Using postural drainage & thoracic percussion to

stimulate production of acceptable sputum. Aerosol induced specimen: Breathing of aerosolized droplets of a solution

containing 15% NaCl and 10% glycerin for approximately 10 min. or until a strong cough reflex is initiated.

No pre-screening required (Although these specimen appear watery resembling saliva but they contain material directly from alveolar spaces ).

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SPECIMEN COLLECTION AND TRANSPORT

3. Gastric aspirate: For isolation of Acid-Fast bacilli. Inability to produce sputum. Nasogastric tube is inserted into the stomach and contests are

withdrawn. The relative resistance of mycobacteria to acidity allows them to

remain viable for short period. Acidity of content is neutralized.

B. Endotracheal or Tracheostomy suctions specimens. Collected in Lukens trap. Tracheostomy aspirates should be treated as sputum.

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ENDOTRACHEAL OR TRACHEOSTOMY SUCTIONS SPECIMENS

Fig: Collection of sputum Fig: Lukens Trap

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SPECIMEN COLLECTION AND TRANSPORT

C. Bronchoscopy Fibreoptic devices Bronchial washings Bronchial aspirates Broncoalveolar lavage BAL (100-300ml NS is infused). Protected bronchial brushing samples.

Broncoalveolar lavage (BAL) 100-300ml NS is infused. Estimated that: 1 million alveoli are sampled during this process. Significant correlation : “Between”

Acute bacterial pneumonia and >103-104 bacterial colonies per mililitres of BAL fluid.

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BRONCHOALVEOLAR LAVAGE (BAL)

SPECIMEN ACCEPTABILITY

Microscopic examination of Gram-stained smear Acceptable

<1% of cells present are squamous epithelial cells

Unacceptable>1% of cells present are squamous epithelial cells

Thorpe JE et. al. 1987. Bronchoalveolar lavage for diagnosing acute bacterial pneumonia. J. Infect. Dis. 155:855-861

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SPECIMEN COLLECTION AND TRANSPORT Mini-BAL: Bedside, Non-bronchoscopic. Using metras catheter. 20ml or less saline is infused.

Protected bronchial brushing: Can collect 0.001 to 0.01 ml of material. Material can be suspended in 1 ml of broth dilution with vigorous

vortexing. Inoculate into culture media using 0.001 ml calibrated inoculating

loop. Correlation to infection: ≥ 1000 CFU/ml of broth diluent. ≥10^6 CFU/ml of original specimen. Have been considered to correlate with infection.

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SPECIMEN COLLECTION AND TRANSPORT

D. Transtracheal Aspirates(TTA): Reduces the likelihood of contamination by upper respiratory

tract flora. Inserting small plastic catheter into the trachea via a needle

previously inserted through the skin and cricothyroid membrane.

E. Other invasive procedure: Thoracentesis (In pleural empyema). Open lung biopsy.

F. Blood culture: Only 20% of patients requiring hospitalization due to pnuemonia

are Blood culture positive.21

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CRITERIA FOR REJECTING SAMPLES

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Mismatch of information on the label and the request

Inappropriate transport temperatureExcessive delay in transportation Inappropriate transport medium

specimen received in a fixativedry specimensample with questionable relevance

Insufficient quantityLeakage

SPECIMEN PROCESSING

The purulent material which contains most of the relevant pathogens, is usually embeded in clear mucoid secretion.

If homogenised: Every drop & loopful of it will contains some of pathogen. Suitable for quantitative examinations.

Dithiothreitol or Buffered Pancreatin Mix and incubate equal volumes of the sputum and a solution of

dithiothreitol or buffered pancreatin.

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SPECIMEN PROCESSING

I. Dithiothreitol Mix rapidly on a vortex mixer for 15 seconds and stand for 15

minutes at ambient temperature. Or mix gently and continuously on a machine that tilts to & fro

placed for 30 minutes in an incubator at 37ºC.

I. Pancreatin Incubate for 30 min. at 37ºC with continuous or occasional

shaking.

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MICROSCOPY

▓ Gram’s stain ▓10 % KOH ▓Calcofluor white stain ▓PASstain.

Fig: Gram’s stain of sputum

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(LPF)

MICROSCOPY

Screening by Gram’s stain Acceptable criteria: <10 epithelial cells /LPF. >25 WBCs/LPF ( Except in leucopenic patients).

Reasonable rejection criteria: >10 epithelial cells /LPF.

Rejection criteria for ETA: >10 epithelial cells /LPF. Or no organism seen under oil immersion.

In Legionella pneumophila Should not be subjected to screening by gram’s stain since sputum may

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MICROSCOPY

27Fig:Neutrophils in gram’s stain Fig: Artifacts in gram’s stain

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CALCULATION OF COMPOSITE Q-SCORE

Use a specimen with a Composite Q score of +1, +2 or +3 for the additional tests. Tests are performed on a specimen with a Composite Q score of <+1 only if specifically ordered by the physician.

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INOCULATION:

Inoculated onto MA and BA at 37ºC. CA at 37ºC in increased carbondioxide condition. 5ug optochin (Ethyl hydrocupriene dihydrocloride to screen

for S. pneumoniae. 5-20 Unit (10U) Bacitracin for H. influenzae. Only specimens obtained by percutaneous aspiration

(including transtracheal aspiration) and by protected bronchial brush are suitable for anaerobic culture.

Buffered charcoal-yeast extract (BCYE) agar for Legionella spp.

PC 0r OFPBL agars for B. cepacia.

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PROCESSING SPECIMENS FOR CULTURE

Process specimens in biological safety cabinet, as aerosol can result in laboratory-squired respiratory infections.

Process all specimens as rapidly as possible, especially specimen from emergency department, and inpatients. Select the most purulent or most blood-tinged portion of the specimen. Significant growth above the cutoff should be reported; however if more than one pathogen is isolated than it is suggestive of oropharyngeal contamination and clinical correlation should be done before reporting the samples.

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CONTAMINATION WITH ORAL FLORA INTERFERES

RESULTS Because of contaminating oral flora ,sputum

specimens, specimens obtained by bronchial washing, and lavage tracheostomy, or endotracheal tube aspirates are not inoculated to enriched broth or incubated anaerobically. Only specimens obtained by percutaneous aspiration (including trans tracheal aspiration )and by protected bronchial brush are suitable for anaerobic culture: he latter must be done quantitatively for proper interpretation.

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IDENTIFICATION OF MICRORGANISM

Identification of microorganism is based on the gram’s staining, colonial characters, biochemical tests and specific anti-sera if required.

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OTHER SPECIMENS

Urine for antigen detection

Blood for serology

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OPTOCHIN SENSITIVITY TEST

“DRAUGHTSMAN” APPEARANCE

BILE SOLUBILITY TEST

LABORATORY DIAGONOSIS

COLONY PROCEDURE /Rapid presumptive test-This method works well on large or mucoid colonies-Select a well-isolated single colony from a blood or chocolate agar plate.-Circle the colony on the bottom of the Petri dish to locate- Place one drop of 2% sodium deoxycholate directly on the colony. -Incubate at 37°Cfor up to 30 minutes. Do not invert the plate. The lid may be left slightly ajar to aid evaporation- When the reagent has dried examine the area for lysis or disintegration of the original colony

Positive result: Colony lysed or disintegrated Negative result: No change

QUELLUNG REACTION (NEUFELD QUELLUNG RXN)

Word origin: German quellung for "swelling" quellung phenomenon

Increase in opacity and visibility of the capsule of capsulated organisms exposed to specific agglutinating anticapsular antibodies

Apparent swelling of the capsule upon binding of homologous antibody due to change in refractive index

reagent - antipneumoccal rabbit sera (omniserum)is used against 90 sero- types of S.pnuemoniae from State Serum Institute, DK2300,Copenhagen, Denmark)

SEROTYPING

different serotypes causes different diseases• Serotyping can be done by:

- Quellung reaction(described by Neufeld in 1902), classical method Agglutination of the pneumococci with type specific antiserum Capillary Precipitation of SSS with type specific antiserum Coagglutination dot blot assay counterimmuno-electrophoresis (CIEP)

Properties Pneumococcus Viridans streptococci

Morphology Capsulated,lanceolate-shaped diplococci

Non-capsulated,oval or round cells in short chains

Colony on BA Raised/flat initially later “draughtsman”

Dome-shaped

Broth culture Uniform turbidity granular with powdery deposit

Bile solubility test + -

Optochin sensitivity Sensitive Resistant

Inulin fermentation + -

Quellung reaction + -

Mice/rabbbit pathogenicity Pathogenic Non -pathogenic

NUCLEIC ACID TECHNIQUE

Polymerase chain reaction Samples : blood, serum, CSF and respiratory samples,

body fluids Gene used as primers

The pneumolysin gene, pneumococcal surface adhesin protein (PsaA) gene, cell wall autolysin (LytA) protein gene

DNA/DNA hybridization

CASE STUDY…….

A 16-month-old boy was admitted with fever, lethargy and trouble breathing. A diagnosis of pneumonia was made by physical examination. The child had recently been to Panama and at that time was treated with ceftriaxone for cough and fever. His fever continued, despite treatment. On admission, he was given erythromycin therapy. Thacheal aspirate and blood cultures were obtained, but the respiratory specimen contained numerous epithelial cells and yielded normal respiratory flora on culture. A pleural aspirate and blood cultures were positive with Streptococcus pneumoniae, which was reisistant to erythromycin and penicillin and intermediate in susceptibility to ceftriaxone. The patient was given high doses of ceftriaxone and vancomycin and responded to therapy. 47

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QUESTIONS..??? What criteria are used in laboratories to reject sputum and

tracheal aspirates for culture?

If greater than 10 squamous epithelial cells per low-power field are seen in a gram stain but the smear also has numerous white blood cells (greater than 25/LPF), should the specimen be rejected for culture?

In cases of pneumococcal pneumonia, what percentage of blood and sputum cultures is positive for S. pneumoniae?

The organism was reported as resistant to penicillin and intermediate in susceptibility to third-generation cephalosporins . How does the laboratory perform testing for this organism?

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SPECIAL CONSIDERATION TO PULMONARY TUBERCULOSIS

Recovery of mycobacteria from clinical samples is more time consuming procedure than for normal pathogenic bacteria, requiring as it does the following steps:

1. Homogenization2. Centrifugation3. Neutralization4. Inoculation5. Incubation for at least 6wks

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CULTURAL CHARACTERISTICS (M. TUBERCULOSIS) M. tuberculosis is an obligate aerobe while M.

bovis is microaerophilic on primary isolation (aerobic on subculture)

Grows slowly (G.T. 15-20 hours) Optimum temperature 370C pH 6.4-7.0 Does not grow <250C and >400C Increased carbondioxide (5-10%) enhances

growth Grows more luxuriantly in culture (eugonic)

than M. bovis (dysgonic)

Addition of 0.5% glycerol improves the growth of M. tuberculosis but has no effect on or even impair the growth of M. bovis.

Sodium pyruvate helps the growth of both types

Human tubercle bacilli do not grow in presence of p-nitrobenzoic acid (500 mg/l), unlike other slow growing nonchromogens.

Highly susceptible even to traces of toxic substance like fatty acid in culture media which can be neutralized by serum albumin or charcoal.

CULTURE MEDIA Solid media:

Egg containing – Lowenstein-Jensen mediaPetragnani mediaDorset egg mediaOgawa media

Blood containing – Tarshis media Serum containing – Loeffler’s serum slope Potato containing – Pawlowsky’s media

Liquid media: Dubo’s media Middlebrook’s media 7H9 Proskauer and Beck’s media Sula’s and Sauton’s media Kirchner media

To prevent overgrowth by contaminants, Cocktail of antibiotics

Polymyxin BAmphotericin BNalidixic acidTrimethoprimAzlocillin

PANTA

Polymyxin B Amphotericin BCarbenicillinTrimethoprim

PACT

COLONY CHARACTERS Solid media:

M. tuberculosis: Colonies appear after 2-3 weeks.

Colonies first appear as small, dry, friable, rough and granular, creamy white (Rough, tough, buff)

Typical colonies have a flat irregular margin and look like a cauliflower in the center.

M. bovis: Colonies appear after 3-6 weeks

Colonies are smooth, translucent and pyramidal

QUANTIFICATION SCALE FOR MYCOBACTERIAL GROWTH ON AGAR PLATES (ATS, 2000)

No. of colonies seen Quantity reported

No colonies seen

<50 colonies

50-100 colonies

100-200 colonies

200-500 colonies (almost confluence)

>500 colonies (confluence)

Negative

Report actual number seen

1 +

2 +

3 +

4+

Liquid media:Bacilli grow either on surface as pellicle or as floccules

throughout the medium due to hydrophobic nature of their cell wall lipid.

Generally, virulent strains often grow as twisted rope like colonies called serpentine cords??????

Diffuse bacterial growth is obtained by adding a detergent Tween 80.

Lowenstein-Jensen media Fresh whole eggs Defined salts Glycerol Potato flour Asparagine Malachite green

Middlebrook 7H10Defined saltsVitaminsCofactorsGlycerolOleic acidAlbuminDextroseCatalaseMalachite green

Ogawa media Potassium dihydrogen phosphate Sodium glutamate Glycerine 2% malachite green Distilled water Whole egg homogenate

Niacin test- Niacin test is positive with human type and negative with bovine type of bacilli.

Aryl sulphatase test- Aryl sulphatase test is positive with atypical mycobacteria.

Neutral red test- Neutral red test is positive with virulent strains of tubercle bacilli while avirulent strains are negative.

Catalase-peroxidase test- Atypical mycobacterial strains - strongly catalase

positive Tubercle bacilli - only weakly positive for catalase Tubercle bacilli - peroxidase positive Atypical mycobacteria – peroxidase negative Catalase and peroxidase activity are lost when tubercle

bacilli become INH resistant.

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Amidase test- Amidase test differentiates mycobacteria by its ability to split amides. A useful pattern is provided by testing five amides, acetamide, benzamide, carbamide, nicotinamide and pyrazinamide.

Nitrate reduction test- M. tuberculosis + M. bovis -

LABORATORY DIAGNOSIS

Sample is collected according to the site of infection

Primary tuberculosis- 3 early morning sputum samples, laryngeal swab, bronchial washings, stomach washings in children

Transport medium for preserving M. tuberculosis in sputum specimens – Cetylpyridinium chloride – Sodium chloride (CPC-NaCl) transport medium.

Conventional techniques Sputum microscopy

Acid fast stains: Ziehl Neelsen stain Kinyoun’s modification of ZN stain Auramine-Rhodamine stain

Sputum cultureMantoux skin test

Direct methodsSmear CulturePhage based assaysPhenotypic methods- lipid analysisMolecular methods- Probes, PCR

Indirect methodsSerology- ELISA, Latex agglutinationELISPOT AssaysDemonstration of biological products

ADA estimation Tuberculostearic acid test Bromide partition test

SPUTUM SMEAR EXAMINATION

Most widely applicable, most reliable ( WHO )

Minimum 3 samples (spot- early morning-spot)

Sensitivity- 50-70%

DISADVANTAGES OF SMEAR EXAMINATION

Lower limit of detection - 104 bacilli/ml

Sample

Cannot determine viability

Cannot identify species

False positive/ False negative result

INTERNATIONAL UNION AGAINST TUBERCULOSIS (IUAT)

If definite pink bacilli are not seen – AFB not found

If 1 or 2 bacilli in the entire field – Doubtful, repeat for another sample

If 1-9 AFB/100 fields – AFB found (exact number)

10-100 AFB/ 100 fields – AFB found (+)

1-10 AFB/ field – AFB found (++)

> 10 AFB/ field – AFB found (+++)

DIGESTION, CONCENTRATION AND CULTURAL METHODS Homogenization – To release the mycobacteria

from the body fluid or tissue in which they are contained

Decontamination – To kill the contaminants or else they will overgrow the medium

1. Petroff’s methodEqual vol. of Sputum and 4% NaOH370C – 30 mins with occasional shakingCentrifugation 3000g – 20-30minsDiscard the supernatantNeutralize the deposit with 8% HCl in presence of

phenol red indicatorCentrifugationDeposit

Inoculated on to the culture media Smear preparation Animal inoculation Molecular methods

2. Modified Petroff’s method Sputum + Double vol. of 4% NaOH R.T. – 15mins with occasional shaking Centrifugation 3000g – 15mins Discard the supernatant 15 ml saline or water + sediment Centrifugation 3000g – 15mins Deposit – inoculated on to the culture media

3. N-acetyl-L-Cysteine-NaOH method

4. Zephiran (Benzalkonium chloride)-TSP method

ANIMAL INOCULATION Intramuscular inoculation of 0.5 ml bacterial suspension into the

thigh of 12 week old guinea pig The tuberculin test becomes positive after 3-4 weeks and there

will be progressive loss of weight of the animal The animal is autopsied after 6wks or 12 wks. Results:

Caseous lesion at the site of inoculation Enlarged inguinal LN Tubercules in the lungs Splenomegaly

The identity of the bacteria is confirmed by demonstration of AFB from the lesions to exclude the Y. pseudotuberculosis, Brucella as their lesions macroscopically simulate tubercles.

ROOT INDEX OF VIRULENCE

CDC CLASSIFICATION OF TUBERCULIN REACTION

An induration (palpable raised hardened area of skin) of more than 5–15 mm (depending upon the person's risk factors) to 10 Mantoux units is considered a positive result, indicating TB infection.

5 mm or more is positive in HIV-positive person,

Patients with organ transplants and other immunosuppressed patients

10 mm or more is positive in Recent arrivals (less than 5 years) from high-prevalent countriesResidents and employees of high-risk congregate settings (e.g.,

prisons, nursing homes, hospitals, homeless shelters, etc.)Persons with clinical conditions that place them at high risk (e.g.,

diabetes, prolonged corticosteroid therapy, leukemia, end-stage renal disease, chronic malabsorption syndromes, low body weight, etc)

15 mm or more is positive in Persons with no known risk factors for TB

Recent advances Serological diagnosis

IgM and IgG antibody to mycobacterial antigens (ELISA, LA) IgG and IgA antibody against the mycobacterial antigen A60 in

patient with EPT

Radiometric BACTEC 460 TB method

MGIT 960 Mycobacterial detection system

Chemical detection of biological compounds Adenosine deaminase Tuberculostearic acid test Bromide partition test HPLC

Mycobacteriophage typing

Genotyping methods

BACTEC Organisms multiply in the broth and metabolize

14C – containing palmitic acid substrate producing radioactively labelled 14CO2 in the atmosphere that collects above the broth in the bottle.

The BACTEC instrument withdraws the 14CO2 – containing atmosphere and measures the amount of radioactivity present which is converted proportionally to a quantitative growth index (GI).

BACTEC MGIT-960

-It is based on a glass tube containing a modified Middlebrook 7H9 broth enriched with OADC and PANTA antibiotic mixture.

-A fluorescent compound is embedded at the bottom of tube.

- The fluorescent compound does not fluoresce in the presence of oxygen, but it fluoresces following depletion of oxygen by actively respiring organisms as a result of mycobacterial growth.

Mycobacterial growth indicator tube (MGIT) system

CULTURE METHODS

Modern method Conventional

Detection 14 days 28 days

Susceptibility 3 wks 8 wks

Sensitivity 95% 84%

Contamination 4% 5%

LYSIS-CENTRIFUGATION BLOODCULTURE SYSTEM

- The recovery of mycobacterium from peripheral blood and bone marrow samples may be improved by releasing the intracellular mycobacterial cells into the blood culture broth, increasing the rate and reducing the time of recovery.

- In the lysis- centrifugation blood culture method, blood is put into a tube containing an anticoagulant and a lysing agent to effect rupture of both erythrocytes and neutrophils.

- Following centrifugation of the tube, the sediment is inoculated into the appropriate culture media.

- This method has increased both the yield and shortened the time of recovery of mycobacteria from blood cultures.

LIPID ANALYSIS

Mycolic acid profiles

HPLC- Confirmation of isolate , directly

from clinical specimen

OTHER TESTS Immunomagnetic separation of M. tuberculosis

(Murtagh et al 1995)

Magnetic polystrene microspheres coated with antibody to mycobacteria are added to the specimen and mixed together

The beads and bound organisms are collected by using a magnet and detected by microscopic examination with the use of conventional method.

It improves mycobacterial detection level by 2-3 times more than conventional method.

Used for CSF, Sputum.

Mycodot antibody test Purified lipoarabinomannan is used as an antigen

and it detects antimycobacterial antibody levels likely to be found in those with active diseases

Specificity 97-98% Sensitivity 70-75%

URINARY ANTIGEN DETECTION

LAM antigen

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MOLECULAR DIAGNOSIS OF M. TUBERCULOSIS

Nucleic acid probes

Amplification techniques

PCR assay

Transcription mediated Assay

Strand displacement amplification

Q β replicase gene amplification

Spoligotyping

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DNA MICROARRAY

NUCLEIC ACID PROBES

DNA probes M. tuberculosis complex, M. avium

Not sensitive enough (104 organisms)

Ribosomal RNA based probes Target r- RNA

M. tuberculosis , M. avium, M. gordonae

Chemiluminiscent system

PCR ASSAYS

Target

IS 6110 (10-20 times repeated in genome) 25% of Indian population lack IS 6110

PCR ( ROCHE AMPLICOR)

Target gene 16S-rRNA (584 bp)

Reproducible, sensitive, specific

Can detect 1-10 organisms

DRAWBACKS OF PCR ASSAY

Dead bacilli are detected.

False Positive due to contamination.

No prognostic value.

DEMONSTRATION OF BIOLOGICAL PRODUCTS

Adenosine Deaminase Assay

Lymphocytic enzyme - Increase in TB

Surrogate marker for extrapulmonary

tuberculosis in Pleural, pericardial, CSF

Sensitivity & specificity > 90%

Limitations - False +

ADA TEST

FAST PLAQUE ASSAY

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