“The renewals of the TB Laboratory Diagnosis”

Nurhan Albayrak (Clinical Microbiologist)April 3, 2013 / Antalya

Outline

Flourecence microscopyMolecular tests for TB identification Moleculer tests for drug susceptibility

testing

TB diagnosis Bacteriological

Smear microscopy Culture (gold standart) Molecular tests Serological tests Interferon gama releasing assay

Smear microscopy Because of the mycobacterial cell wall

lipit content Late culture positivity (4-8 week) Difficulty to stainig

mikolik acids carbons are so much

Smear microscopy Advantages

Simple, cheap, fast (<1 hour) Abundant bacilli in cavitary disease

Disadvantages Sensitivity 25-80% (5.000 bacil/ml) Less sensitivity in extrapulmonary TB and NTM Needs experience (10-15 slides/week) Maximum capacity for one person is 25 slides/daily

Deficiency Didn’t disriminate M. tuberculosis complex from NTM Didn’t disriminate viable bacteria from the dead one

Renewal LED (light emitting diode)

Compared with EZN staining (patient number is 2.355) Sensitivity 2 sputum LED %68,8-76,5, EZN %61,6-69,8 3 sputum LED %73,3-80,6, EZN %66,4-74,4

Specivicity 2 sputum LED %89,5-92,2, EZN %97,3-98,6 3 sputum LED %86,5-89,6, EZN %96,8-98,2

Cuevas LE, et al. PLoS Med. 2011

Smear microscopyWhat’s the advantages of

LED? Sensitivity↑ Capacity↑ Need to lab staff ↓

Is there any clinical advantage?

Interpretation of the results

What means AFB (+) results?a) Acid fast bacilli positiveb) Mycobacteria positivec) M. tuberculosis complex positived) M. tuberculosis positive

What means AFB (-) results? This results don’t means that ‘the patient is not TB’

Because the sensitivity is 25-80%

Molecular tests For detection

PCR Real-time PCR Microarray

For identification For DST

Real-time PCR Line probe assay Sequencing

Molecular tests Based on the principle of amplification of the

M. tuberculosis DNA

AdvantagesSensitivity changed according to the method (10-

100%)Fast

DisadvantagesExpensive

DeficiencyDidn’t disriminate viable bacteria from the dead oneDidn’t validated for extrapulmonary TB

Metaanalysis with 45

investigation In pulmonary TB9-100%

sensitivity25-100%

specitivity

SensitivitySpecivicity

Sarmiento OJ, et al. J Clin Microbiol. 2009

Renewal in molecular tests Xpert Mtb/RIF assay

Detection of Mtb and RIF resistance Handling from sputum Resulted in 2.5 hours

Xpert Metaanalysis with 18 investigation, 10244

sample In pulmonary TB; sensitivity 90.4%, specivity 98.4%Detection limit; 131 CFU/ml For RIF resistance; sensitivity 94.1%, specivity 97%

Chang K, et al. J Infect. 2012

WHO’s recommendations for Xpert (2011)

In MDR-TB suspected cases Means RIF resistance MDR? RIF resistance is an indicator for MDR

Smear negative cases Sensitivity is 70%

Drug susceptibility testing Conventional = phenotypic methods

LJ / Agar proportion Automated liquid cultures

Molecular = genotypic methods Detecting the mutation

Real-time PCR Line probe assay Sequencing

Advantages - disadvantagesMolecular methodsDetecting mutaion DNA is enough High in mutation

Fast from sputum (1-7 days)

ExpensiveNeed experienceNeed equipmentBSL-2Need verification

Conventional methods

Growth inhibitionNeeds viable bacteriaHigh fot HRFor second line drugs ↓ Late from culture (after

culture 4-35 days)Cheaper than molecular testsSimple than molecular testsLess equipmentBSL-3

Principle

Sensitivity

Turn-on time

Technic

BiosafetyDeficiency

Mutation regions related with resistance

%40-60%25%10%95%95%60%20

%60%80-90%90

Silva PEA, et al. J Antimicrobial Chemother. 2011

Line probe assay

AFB (-/+) sample; HR resistance AFB (+) samle; EMB, FLQ and AG/CP

resistanceSteps

DNA extraktion (45 min) Amplification (2-3 h) Hybridisatioon (2-3 h)

Line probe assay (sL)

Interpretation of the resultsReal R False (+) False (-)

RIF resistance 180 4 (2,2%)* 1 (0,5%)(rpoB mutation) INH resistance 391 2 (0,5%) 43 (10,9%)(katG / inhA mutation) EMB resistance 120 13 (10,8%)

23(19,1%)(embB mutation) FLQ resistance 13 0 1 (7,6%)(gyrA mutation) AG/CP resistance 12 1 (8,3%) 10 (83%)(rrs mutation)

*Any mutation with wild type missings

Summary The new molecular tests

recommended in identification of pulmonary TB cases

Using molecular tests for DST Is faster for MDR detection Recommended for some situations Don’t forget that the tests need to

verification with convantional methods.

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