“ the renewals of the tb laboratory diagnosis ”
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“ The renewals of the TB Laboratory Diagnosis ”. Nurhan Albayrak ( Clinical Microbiologist ) April 3, 2013 / Antalya. Outline. Flourecence microscopy Molecular tests for TB identification Moleculer tests for drug susceptibility testing. TB diagnosis. Bacteriological - PowerPoint PPT PresentationTRANSCRIPT
“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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