DIAGNOSIS OF TUBERCULOSIS Dr T V Rao

8/14/2019 DIAGNOSIS OF TUBERCULOSIS Dr T V Rao

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Dr.T.V.RaoDr.T.V.Rao

Robert Koch DiscoversRobert Koch Discovers

MycobacteriumMycobacterium



Dr.T.V.Rao

A Global EmergencyA Global Emergency

The Tuberculosis in theThe Tuberculosis in the

beginning of the 21beginning of the 21stst

CenturyCenturydeclared as Global Emergencydeclared as Global Emergency

(WHO)(WHO)




Why Tuberculosis is a ImportantWhy Tuberculosis is a Important

Disease.Disease. Tuberculosis continues to be aTuberculosis continues to be a

Important communicable disease.Important communicable disease. A leading cause of morbidity andA leading cause of morbidity and

mortality in Developing world.mortality in Developing world. Most Important communicableMost Important communicable

disease in Bangladesh, China,disease in Bangladesh, China,

Indonesia, Africa, and Pakistan.Indonesia, Africa, and Pakistan.

But it is Curable DiseaseBut it is Curable Disease




Tuberculosis is a Global ProblemTuberculosis is a Global Problem




Tuberculosis - ImportantTuberculosis - Important

communicable disease spread bycommunicable disease spread by

Respiratory routeRespiratory route




Why Everybody Concerned.Why Everybody Concerned.

Tuberculosis kills young adults.Tuberculosis kills young adults.

Premature death of the infected aPremature death of the infected a

prominent future.prominent future. Today many are co infected with HIV.Today many are co infected with HIV.

The open cases of Tuberculosis infects aThe open cases of Tuberculosis infects a

few around his/her environment.few around his/her environment. A social burden to the family, society andA social burden to the family, society and

Nations.Nations.




Tuberculosis in the era of Tuberculosis in the era of

HIV / AIDS.HIV / AIDS.

HIV / AIDS epidemic led to largeHIV / AIDS epidemic led to large

increase of Smear negativeincrease of Smear negative

pulmonary tuberculosis which in turnpulmonary tuberculosis which in turn

has led to poor treatment out comes,has led to poor treatment out comes,

and early mortalityand early mortality

Frequently involves Lower lobes of Frequently involves Lower lobes of

Lungs.Lungs.




Why we fail to DiagnoseWhy we fail to Diagnose

Tuberculosis.Tuberculosis.

Lack of health infrastructure.Lack of health infrastructure.

Control is plagued with lack of Control is plagued with lack of

Accurate,Accurate,

Robust,Robust,

and Rapidand Rapid

Diagnostic methods,Diagnostic methods,

Technologies.Technologies.




Why we failed ( Cont )Why we failed ( Cont )

Diagnostic services are poor, and soDiagnostic services are poor, and so

we failed at Individual andwe failed at Individual and

community levels.community levels.

Patients are diagnosed late.Patients are diagnosed late.

Many patients are never diagnosedMany patients are never diagnosedbefore death.before death.

Early deaths are burden toEarly deaths are burden to

Social Infrastructure andSocial Infrastructure andEconomic loss.




Importance of Clinical servicesImportance of Clinical services

Early diagnosis rests with clinicians,Early diagnosis rests with clinicians,

whose contribution is immense inwhose contribution is immense in

prompt treatment.prompt treatment.

A clinicians knowledge, properA clinicians knowledge, properdocumentation are immense help indocumentation are immense help in

Developing countries.Developing countries.




When to suspect TuberculosisWhen to suspect Tuberculosis

Cough longer than 3 weeks.Cough longer than 3 weeks.

Fever for 1 month, or both.Fever for 1 month, or both.

Blood stained sputum.Blood stained sputum. Night sweats, weight lossNight sweats, weight loss Age between 14 and 70 yearsAge between 14 and 70 years

(( Correlates National TuberculosisCorrelates National TuberculosisProgramme ).Programme ).



Dr.T.V.Rao

DIAGNOSTIC METHODSDIAGNOSTIC METHODS




Diagnosis.Diagnosis.

Tuberculosis is a diversified disease.Tuberculosis is a diversified disease.

Any organs can be involved.Any organs can be involved.

Any age group, gender no bar forAny age group, gender no bar for


Involvement of Lungs contribute toInvolvement of Lungs contribute to

majority of tuberculosis.majority of tuberculosis. And involvement of Lungs is designated asAnd involvement of Lungs is designated as

Pulmonary tuberculosis.Pulmonary tuberculosis.




Diagnosis of PulmonaryDiagnosis of Pulmonary

TuberculosisTuberculosis

Majority of Adults suffer with pulmonaryMajority of Adults suffer with pulmonarytuberculosis.tuberculosis.

Microbiological examination of SputumMicrobiological examination of Sputumcontinues to be a Gold standard in provingcontinues to be a Gold standard in provingthe Diagnosis.the Diagnosis.

Sputum examination in Children is notSputum examination in Children is not

sensitive in Diagnosis.sensitive in Diagnosis. Radiological examination of Lungs, mostRadiological examination of Lungs, most

commonly prescribed investigation.commonly prescribed investigation.




X - ray examination of chest mostX - ray examination of chest most

easily available Investigation.easily available Investigation.



Dr.T.V.Rao

MicrobiologicalMicrobiologicalInvestigations are essentialInvestigations are essential

for definitive Diagnosis of for definitive Diagnosis of Tuberculosis.Tuberculosis.




Importance of Optimal SpecimensImportance of Optimal Specimens

Pulmonary Tuberculosis is thePulmonary Tuberculosis is the

commonest presentation of commonest presentation of


Sputum is the Most importantSputum is the Most important

specimen for identification andspecimen for identification and

isolation of Acid fast bacilli.isolation of Acid fast bacilli.

The developing countries suffers theThe developing countries suffers the

most important step in getting anmost important step in getting an

ideal sample.ideal sample.



Dr.T.V.Rao

Sputum specimensSputum specimens

Train the staff to obtain the appropriateTrain the staff to obtain the appropriatespecimenspecimen

A few minutes of education to patients onA few minutes of education to patients on

importance of ideal sample make a greatimportance of ideal sample make a greatdifference and improves the Diagnosis.difference and improves the Diagnosis.




Observe to identify Sputum fromObserve to identify Sputum from

Saliva.Saliva.

SPUTUMSPUTUM

Specimens appear mucoidSpecimens appear mucoideven, blood stained.even, blood stained.

Contains manyContains many

Polymorphoneutrophils.Polymorphoneutrophils.

SALIVASALIVA

Appears clear, watery,Appears clear, watery,and frothy.and frothy.

Contains manyContains many

squamous epithelialsquamous epithelial

cellscellsAbsence of Absence of

Polymorphoneutrophils.Polymorphoneutrophils.




Role of Microscopy in Tuberculosis.Role of Microscopy in Tuberculosis.

Microscopy for Diagnosis of Tuberculosis isMicroscopy for Diagnosis of Tuberculosis is

initiated in 1880initiated in 1880 The conceptions have not changed sinceThe conceptions have not changed since

then.then. Best efforts should be put to obtainBest efforts should be put to obtain

sputum,sputum, Processing of saliva loses all valuable cluesProcessing of saliva loses all valuable clues

to diagnose.to diagnose.




Microscopy and TuberculosisMicroscopy and Tuberculosis

Microscopy withMicroscopy with

Ziehl – Neelsen’sZiehl – Neelsen’s

stainingstaining

A century oldA century old

procedureprocedure




Why MicroscopyWhy Microscopy

Only we need Microscope, and fewOnly we need Microscope, and few

stains.stains.

Most rapid, economical,Most rapid, economical,

Can detect bacterial load.Can detect bacterial load.

A Diagnostic, and Prognostic tool.A Diagnostic, and Prognostic tool.

A little of sputum 0.2 µl is adequate.A little of sputum 0.2 µl is adequate. A prompt diagnosis after searchingA prompt diagnosis after searching

as few as 100 fields.as few as 100 fields.




Limitation of Microscopy for Limitation of Microscopy for


Repeated sample examinations. load onRepeated sample examinations. load ontechnical staff.technical staff.

Training and dedication of Microscopist.Training and dedication of Microscopist. The load of bacilli must be more thanThe load of bacilli must be more than

10,000 / 1 ml of sputum.10,000 / 1 ml of sputum. Low in sensitivity < 50 %Low in sensitivity < 50 % Repeated requests for samplesRepeated requests for samples High drop out by patients, for repeatedHigh drop out by patients, for repeated

samples.samples. Not dependable in pediatric age group.Not dependable in pediatric age group.




Smear showing Acid Fast Bacilli.Smear showing Acid Fast Bacilli.




What is Smear PositivityWhat is Smear Positivity

WHOWHO

All patients who have submittedAll patients who have submitted

twotwo

Specimens and found to beSpecimens and found to bepositivepositive

for identification of AFBfor identification of AFB




Processing Direct smear negativeProcessing Direct smear negative

specimensspecimens Sputum Microscopy can be improved withSputum Microscopy can be improved with

Sputum liquefaction, concentration andSputum liquefaction, concentration andgravity sedimentation.gravity sedimentation.

Popular solventsPopular solventsSodium hypochlorite.Sodium hypochlorite.

Sodium hydroxide.Sodium hydroxide.

Ammonium sulphateAmmonium sulphate

N-acetyl-L-cysteine –sodiumN-acetyl-L-cysteine –sodiumhydroxide.hydroxide.




Benefits of Liquefaction andBenefits of Liquefaction and

ConcentrationConcentration

Major studies showed processing of Major studies showed processing of sputum with chemicals and centrifugationsputum with chemicals and centrifugation

improved sensitivity up to 18 %.improved sensitivity up to 18 %. Incremental yield ( positive with bleachIncremental yield ( positive with bleachminus positives with Ziehl – Neelsen stain)minus positives with Ziehl – Neelsen stain)up to 9 %.up to 9 %.

Treating specimens with SodiumTreating specimens with Sodiumhypochlorite is Mycobactericidal and alsohypochlorite is Mycobactericidal and alsokills HIV and improves the safety andkills HIV and improves the safety andacceptability by technical staff.acceptability by technical staff.




When Microscopy failsWhen Microscopy fails

Smear negative tuberculosis.Smear negative tuberculosis. In HIV infected patients, on manyIn HIV infected patients, on many

occasions prove negative. in spite of occasions prove negative. in spite of

presence of bacilli, ( as few bacilli arepresence of bacilli, ( as few bacilli areexpectorated).expectorated).

Needs concentration and liquefactionNeeds concentration and liquefaction

with chemicals.with chemicals. Time consuming, needs moreTime consuming, needs more

technical manpowertechnical manpower




Growing role of Growing role of

Fluorescent MicroscopyFluorescent Microscopy

There is a growing need for screening forThere is a growing need for screening for

AFB by Florescent Microscopy.AFB by Florescent Microscopy. Several studies prove, FlorescentSeveral studies prove, Florescent

Microscopy in Diagnosis of Tuberculosis isMicroscopy in Diagnosis of Tuberculosis is

a priority,a priority, Developing world should opt and initiateDeveloping world should opt and initiate

florescent microscopy.florescent microscopy.




Acid Fast Bacilli as seen under Acid Fast Bacilli as seen under

Fluorescent MicroscopeFluorescent Microscope




Why we need FlorescentWhy we need Florescent

MicroscopyMicroscopy Useful when few bacilli are present.Useful when few bacilli are present. Increases the sensitivity in HIV patients withIncreases the sensitivity in HIV patients with

tuberculosis.tuberculosis. Reduces the time needed for testing.Reduces the time needed for testing. About 15 times as many fields of view can beAbout 15 times as many fields of view can be

scanned by fluorescent microscopy than by Ziehlscanned by fluorescent microscopy than by Ziehl– Neelsen’method in the same period.– Neelsen’method in the same period.

Increases the sensitivity by 10 %Increases the sensitivity by 10 %

Better conclusions with one or two specimens,Better conclusions with one or two specimens,unlike Ziehl Neelsen’s method needing 3 or > 3unlike Ziehl Neelsen’s method needing 3 or > 3specimens.specimens.




Culturing MycobacteriumCulturing Mycobacterium

Culturing for isolation of Culturing for isolation of

Mycobacterium spp continues to be aMycobacterium spp continues to be a

Gold standard, particularly inGold standard, particularly in

Developing countries.Developing countries.

Need only 10 – 100 bacilli / 1 ml of Need only 10 – 100 bacilli / 1 ml of

sputum.sputum.




CulturingCulturing

Most useful inMost useful in Surveillance,Surveillance, Drug sensitivity testing patterns.Drug sensitivity testing patterns. Identify treatment failures.Identify treatment failures.

Useful in Patients presenting withUseful in Patients presenting withrespiratory symptoms, X- ray’srespiratory symptoms, X- ray’ssuggestive, but smear negative. Can provesuggestive, but smear negative. Can proveculture positive.culture positive.

Cultures remain suggestive and helpful inCultures remain suggestive and helpful inearly treatment periods, failed drugearly treatment periods, failed drugregimes.regimes.




Methods of Culturing.Methods of Culturing.

Culturing on Lowenstein Jenson’sCulturing on Lowenstein Jenson’s

culture medium remain theculture medium remain the

affordable ,economical method inaffordable ,economical method in

developing world.developing world.




Limitation in CulturingLimitation in Culturing

Mycobacterium spp are slowMycobacterium spp are slow

growing.growing.

Need 6 – 8 weeks for growing.Need 6 – 8 weeks for growing.

Specimens can be contaminatedSpecimens can be contaminated

while growing, needs repeatedwhile growing, needs repeated

specimens, in turn patients loosespecimens, in turn patients loose

confidence in Laboratories.confidence in Laboratories.




Recent facts on CulturingRecent facts on Culturing

Useful in HIV infected patients withUseful in HIV infected patients with


As even few bacilli can be grown inAs even few bacilli can be grown in

spite of smear negativity.spite of smear negativity.

But the specimens to be incubatedBut the specimens to be incubated

for longer time as few bacilli arefor longer time as few bacilli are

present.present.




Pitfalls in CulturingPitfalls in Culturing

Specificity is lost due toSpecificity is lost due tocontamination.contamination.

Can yield false positive results in 1 –Can yield false positive results in 1 –

4 % of the cases.4 % of the cases.

Cultures may be negative in spite of Cultures may be negative in spite of

x rays are suggestive of tuberculosis.x rays are suggestive of tuberculosis.




Growth of Acid fast bacilli onGrowth of Acid fast bacilli on

L J Medium.L J Medium.



Dr.T.V.Rao

ADVANCES INADVANCES IN

CULTURINGCULTURING

TECHNIQUES.TECHNIQUES.There are emerging Modern MediaThere are emerging Modern Media

with accurate detection, arewith accurate detection, are

replacing the Egg and Agar basedreplacing the Egg and Agar basedmedium.medium.




Emerging methods in CulturingEmerging methods in Culturing

MGIT – Mycobacterium growthMGIT – Mycobacterium growth

incubator tube method.incubator tube method.

Growth occurs in shorter than eggGrowth occurs in shorter than egg

medium.medium.

Usefulness in HIV patientsUsefulness in HIV patients

established.established.

Contamination is lessContamination is less

But expensive to people inBut expensive to people in

Developing world.Developing world.




Blood culturing for MycobacteriumBlood culturing for Mycobacterium

Useful in HIV patients, and children.Useful in HIV patients, and children.

Effective in isolation of AtypicalEffective in isolation of Atypicalmycobacterium.mycobacterium.

But not cost effective.But not cost effective.

May be important tool in future forMay be important tool in future fordiagnosing Tuberculosis in HIVdiagnosing Tuberculosis in HIV

infected.infected.



Dr.T.V.Rao

Molecular Methods inMolecular Methods in

Diagnosis of TuberculosisDiagnosis of TuberculosisSeveral methods areSeveral methods are

available, mainly used asavailable, mainly used asResearch toolsResearch tools




Real Time PCR replacing older Real Time PCR replacing older

MethodsMethods




PCR How useful to our Patients?PCR How useful to our Patients?

PCR ( Polymerase chain reaction ) used byPCR ( Polymerase chain reaction ) used by

several investigators.several investigators.

However most cases can be diagnosedHowever most cases can be diagnosedwith simple methods if effectively used.with simple methods if effectively used.

The definite role of PCR continues to beThe definite role of PCR continues to be

controversialcontroversial

Above all not cost effective to DevelopingAbove all not cost effective to Developing

countries.countries.



Dr.T.V.Rao

Rapid Diagnostic MethodsRapid Diagnostic Methods

in Tuberculosisin TuberculosisPast decade has seen severalPast decade has seen several

emerging technologiesemerging technologiesHow far practicable ?How far practicable ?




Emerging Rapid Methods.Emerging Rapid Methods.

1. Fast Plaque TB uses phage amplification1. Fast Plaque TB uses phage amplification

technology.technology.

2. ELISA ( QuantiFERON – TB )2. ELISA ( QuantiFERON – TB )

3. Enzyme-Linked immunospot3. Enzyme-Linked immunospot

( ELISPOT )( ELISPOT )

ELISPOT proved highly useful to detect activeELISPOT proved highly useful to detect active

tuberculosis in Adults and children.tuberculosis in Adults and children.

E i T h l




Emerging TechnologyEmerging Technology

MODSMODS Microscopic observation drug susceptibilityMicroscopic observation drug susceptibility

assay. ( MODS )assay. ( MODS ) A new method gained importance inA new method gained importance in

several reviews.several reviews. Use a tissue culture plate based assayUse a tissue culture plate based assay

with use of Middle Brook 7HG.with use of Middle Brook 7HG. Needs a inverted light microscope.Needs a inverted light microscope.

Even the drug resistance can be testedEven the drug resistance can be testedwith Rifampicin,and Isoniazid.with Rifampicin,and Isoniazid.

Safe to work with cultures.Safe to work with cultures.



Dr.T.V.Rao

Non Specific TestsNon Specific Tests

Tuberculin testTuberculin test

( Mantoux Test )( Mantoux Test )

T b li TT b li T t




Tuberculin TestTuberculin Test

( Mantoux Test )( Mantoux Test )

Test to be interpretedTest to be interpreted

in relation to clinicalin relation to clinical

evaluation.evaluation.

Even the induration of Even the induration of

5 mm to be5 mm to be

considered positiveconsidered positive

when tested on HIVwhen tested on HIV

patients.patients. Lacks specificity.Lacks specificity.




Serology in Tuberculosis.Serology in Tuberculosis.

Several serological methods wereSeveral serological methods were

evaluated.evaluated.

But never gained the acceptance of But never gained the acceptance of

the majority of the clinicians.the majority of the clinicians.

Serological tests are low sensitivity.Serological tests are low sensitivity.

Many physicians depend on serologyMany physicians depend on serology

in extra pulmonary tuberculosis.in extra pulmonary tuberculosis.






HIV/AIDSHIV/AIDS -- TuberculosisTuberculosis

Consider the HIV statusConsider the HIV status Identify the severity of Tuberculosis.Identify the severity of Tuberculosis. Early use of chest radiography.Early use of chest radiography.

Maximal number of sputum smearMaximal number of sputum smearexaminations.examinations.

Sputum concentration methods to beSputum concentration methods to beencouraged even by smaller laboratories.encouraged even by smaller laboratories.

Explore the use of Florescent Microscopy.Explore the use of Florescent Microscopy. All smear negative specimens should beAll smear negative specimens should be

cultured.cultured.




Limitations of Rapid TestsLimitations of Rapid Tests

The testing needs advanced andThe testing needs advanced and

sophisticated infrastructure.sophisticated infrastructure.

These tests are known for theirThese tests are known for their

inability to diagnose between activeinability to diagnose between active

disease and latent infection.disease and latent infection.

Exclusively used in DevelopedExclusively used in Developed

nations.nations.



Dr.T.V.Rao

Extra pulmonaryExtra pulmonary

TuberculosisTuberculosisPoses several challenges, YetPoses several challenges, Yet

no optimal, specific diagnosticno optimal, specific diagnostic

methodsmethods




Extra pulmonary TuberculosisExtra pulmonary Tuberculosis

A real challenge to Clinicians andA real challenge to Clinicians and

Laboratories.Laboratories.

Optimal specimen collection a priority,Optimal specimen collection a priority, Molecular Methods are growing need.Molecular Methods are growing need.

Clinicians start drug regimes on empiricalClinicians start drug regimes on empirical

basis.basis. Several serological tests for antibodySeveral serological tests for antibody

determinations are evaluated.determinations are evaluated.



Dr.T.V.Rao

Identification of AtypicalIdentification of Atypical

MycobacteriumMycobacteriumA growing concern on infections withA growing concern on infections with

less known, uncommon Mycobacteriumless known, uncommon Mycobacteriumin immunosupreesed, an emergingin immunosupreesed, an emerging

infectious disease probleminfectious disease problem




Atypical MycobacteriumAtypical Mycobacterium

Needs the help of referenceNeeds the help of reference

laboratories.laboratories.

Needs different drug regimes, unlikeNeeds different drug regimes, unlike

typical Mycobacterium isolates.typical Mycobacterium isolates.

Now a gowning concern in the era of Now a gowning concern in the era of

AIDS.AIDS.




Future perceptionsFuture perceptions

It is highly essential to explore and discoverIt is highly essential to explore and discoverrapid, simple, and accurate tuberculosisrapid, simple, and accurate tuberculosisdiagnostic tools.diagnostic tools.

A massive investment, greater scientific interest,A massive investment, greater scientific interest,

political commitment a top priority,political commitment a top priority, Man power development, Human resourceMan power development, Human resource

utilization a greater concern.utilization a greater concern. Microscopy and Florescent Microscopy utilizationMicroscopy and Florescent Microscopy utilization

should be immediate concern, and strengtheningshould be immediate concern, and strengthening

of treatment initiation protocols.of treatment initiation protocols. Effective methods in diagnosing smear negativeEffective methods in diagnosing smear negative

patients a growing priority.patients a growing priority.

Mi i T b l iMicroscopy in Tuberculosis




Microscopy in TuberculosisMicroscopy in Tuberculosis

TODAYTODAY

In spite of severalIn spite of several

scientific, andscientific, and

molecularmolecularadvancesadvances

Microscopy inMicroscopy in


continues to becontinues to beback bone inback bone in

Diagnosis.Diagnosis.

Specific detection of activeSpecific detection of active



Dr.T.V.Rao

Specific detection of activeSpecific detection of active

Tuberculosis cases inTuberculosis cases in

patients with HIV infection or patients with HIV infection or AIDS is feasible andAIDS is feasible and

improves the rate of earlyimproves the rate of earlydiagnosis and successfuldiagnosis and successful

treatment of Tuberculosis.treatment of Tuberculosis.






Created for Medical graduates andCreated for Medical graduates and

Health care workers in Developing worldHealth care workers in Developing world

Dr.T.V.Rao, MD.Dr.T.V.Rao, MD.

e mail;e mail; [email protected]@gmail.com

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DIAGNOSIS OF TUBERCULOSIS Dr T V Rao