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1 Diagnosis of TB Disease: Laboratory Ken Jost, BA May 10, 2017 TB Intensive May 9-12, 2017 San Antonio, TX EXCELLENCE EXPERTISE INNOVATION Ken Jost, BA, has the following disclosures to make: No conflict of interests No relevant financial relationships with any commercial companies pertaining to this educational activity 1
2 TB Diagnostic Methods AFB Smear Nucleic Acid Amplification AFB Culture IGRA (Interferon Gamma Release Assay) TST (Tuberculin Skin Test) X-ray Clinical 3 TB Diagnostic Methods Related to # of Bacilli in Sputum # of Pulmonary TB Cases No AFB ~15% 85% 65% 35% ,000 10, ,000 1,000,000 # AFB/ml of sputum Radiography and clinical (~15%) Poor microscopy ~35% of TB cases Excellent microscopy ~ 65% of TB cases Culture (~85%) and PCR (~80%) of TB cases Adapted from Priorities for TB Bacteriology Services in Low-Income Countries, 2007, IUATLD 2
3 DSHS-Austin TB Testing Algorithm Process Sputum Specimen 2 Days 24 hours 2 Days Nucleic Acid Amplification Test - AFB Smear Microscopy Molecular Detection of Rifampin Resistance NAAT (GeneXpert) (GeneXpert) Inoculate MGIT & 7H11 Culture Media 2-6 weeks Species identification -HPLC 2-3 weeks Drug Susceptibility Testing MGIT 960 & 7H10 Agar Proportion 2 Days Molecular Detection of 1 st and 2 nd Line Drug Resistance (CDC MDDR) Specimen Quality Accurate laboratory results are directly related to the quality of the specimen Sputum Recently discharged material from the bronchial tree, with minimal amounts of upper respiratory tract secretions Well coached patient, collect at least 3ml Label tube, form, and indicate test: initial Dx: Smear, NAAT, & Culture Follow-up: Smear and Culture Release from respiratory isolation? Order Smear only Transport to lab cool and quickly 6 3
4 Acid Fast Bacilli Microscopy (AFB Smear) Has many qualities of an ideal diagnostic test Rapid & universally available Detects the most infectious cases Used to support diagnosis and identify need to isolate Helps monitor response to therapy Identifies priority cases for nucleic acid amplification (NAA) Problems Not sensitive - misses ~50% of TB Not specific in low TB prevalence areas (e.g. Texas) Positive smear may be NTM (16% at DSHS-Austin) Highly specific where TB is highly prevalent 7 AFB Smear one microscopic field 8 4
5 Nucleic Acid Amplification Tests (NAAT) Tiny amounts of DNA/RNA are amplified (copied) until there is enough for easy detection DNA/RNA is examined Identification Detection of Drug Resistance Test turnaround time measured in hours 9 Nucleic Acid Amplification Tests (NAAT) Detects M. tuberculosis complex nucleic acids; does not distinguish between live and dead bacilli For initial diagnostic specimens only Not suitable for follow-up specimen or monitoring; cured patients may be NAAT + for years! Sensitivity compared to TB culture >95% for AFB smear-positive Only 55-75% for AFB smear-negative Does not replace culture for bacteriological diagnosis (Yet) 10 5
6 Who Should be Tested? CDC recommends NAAT on the 1 st sputum from every person suspected to have TB for whom the test result would alter case management or TB control activities Definition of a a person suspected to have TB vary among jurisdictions NAAT should NOT be ordered if: Hospital/commercial lab already reported NAAT+ Clinical suspicion is very low, e.g. other Dx probable, spec is to r/o AFB TB programs, clinicians, and laboratorians must collaborate to develop policy and procedure for patients to be tested 11 How Do I Get a NAAT from the State Lab? DSHS automatically performs NAAT on new patient smear positive respiratory specimens 12 6
7 GeneXpert MTBC Detection Performance DSHS-Austin Nov 2012 Jan 2014, 1.7 Years (1,178 sputum specimens) AFB Smear Positive Mtbc Reference Result Positive Negative Total 99% Accuracy Xpert Positive % Sensitivity Xpert Negative % Specificity Total % Prevalence 99% PPV 98% NPV AFB Smear Negative Mtbc Reference Result Positive Negative Total 98% Accuracy Xpert Positive % Sensitivity Xpert Negative % Specificity Total % Prevalence 89% PPV 98% NPV 13 GeneXpert Rifampin Resistance Performance DSHS-Austin Nov 2012 May 2015, 2.4 yr. (4,579 specimens) Rifampin Phenotype Resistant Susceptible Total 99%Accuracy Xpert Rifampin-R %Sensitivity Xpert Rifampin-S %Specificity Total %Prevalence 70%PPV 99.9%NPV 14 7
8 GeneXpert MTB/Rif Performance in Low MDR Prevalence Settings Negative Predictive Value for Rifampin resistance approx. 100% Predicts RIPE is adequate until growth-based drug susceptibilities become available INH-R common INH & EMB-R rare Approx. 5-15% of rifampin resistant predictions are false due to Phe514Phe silent mutation Rifampin resistant results must be confirmed 15 AFB Culture More sensitive than smear 5,000 to 10,000 AFB/ml for smear ~10 viable AFB/ml for culture NAAT negative, culture positive Required for drug susceptibilities & genotype Positive for only ~85% of PTB Requires a quality specimen May be negative due to contamination Culture also used to monitor patient response to treatment Lengthy 1-6 weeks by liquid media weeks by solid media 8
9 AFB Culture Media Two major categories of media Liquid: also often referred to as broth media Used with automated systems 3 systems are FDA-cleared in US: Biomerieux BacT/ALERT 3D Becton Dickinson BACTEC MGIT Thermo Scientific VersaTREK Solid: egg-based and agar-based 7H11 media Most labs use one liquid and one solid LJ media 17 % Detected DSHS-Austin MTBC Culture Positive Time to Detection All (n=5,805) Mtb positive MGIT cultures from 1/1/2010 thru 12/31/ % 90% 90% 95% Specimen 80% Smear Positive 70% All 60% 50% 50% Smear Negative 40% 30% 20% 10% 0% Time to Detection in Days 9
10 AFB Culture Identification GenProbe AccuProbe, aka Probe High Performance Liquid Chromatography (HPLC) Other Lab Developed Tests Some labs use PCR for AFB Culture ID Reports can be confusing!! 19 M. tuberculosis complex All positive by NAAT & AccuProbe Species No. of Texas strains * M. tuberculosis 12,948 (98.0%) M. bovis 191 (1.4%) M. bovis BCG 51 (0.4%) M. africanum 20 (0.2%) M. caprae M. microti M. canettii M. pinnipedii M TB Complex 20 * Data: Texas DSHS Laboratory Genotype Database 10
11 Tuberculosis Genotyping What have been the most useful aspects of universal DNA fingerprinting of MTBC? Detecting previously unrecognized cases of transmission Reactivation/Relapse vs. Reinfection Detecting M. bovis & M. bovis BCG TB program efficacy Detecting false positive cultures 21 Mtb False Positive Cultures Burman & Reves, Clin Infect Dis 2000, 31: False positive are not rare Median false-positive rate = 3.1% [range 2.2%-10.5%] Clerical errors were as common as lab errors Single specimen positive was sensitive, but nonspecific indicator of false + Low colony count (solid medium) Long time to positivity (broth medium) Think possible false + culture Contact lab and request genotype comparison 22 11
12 Sputum Processing for AFB Smear and Culture Courtesy Frances C. Tyrrell, CDC M. tuberculosis Complex DST Current Recommendations Initial isolate should be tested against first-line drugs (FLD) Isoniazid, Rifampin, Ethambutol, Pyrazinamide Repeat DST if patient is cult+ after 3 mo. Rx For isolates resistant to Rifampin or to any 2 FLDs, test second-line drugs Include Fluoroquinolone, Ethionamide, & Injectable (Amikacin, Capreomycin, or Kanamycin) 24 12
13 M. tuberculosis Complex Drug Susceptibility Testing (DST) Susceptibility testing based on ability of isolate to grow in medium containing single critical concentration of drug Critical concentration represents lowest concentration that inhibits 95% of wild strains (never exposed to drug) Resistance = growth of >1% of inoculum in presence of critical concentration of drug 25 Growth-Based Methods for DST MGIT 320 or 960 VersaTREK Indirect Agar Proportion Company Becton Dickinson Thermoscientific N/A Media Liquid broth Liquid broth FDA Cleared Yes Yes Solid medium Petri plate No (laboratory developed test) Time to Results days days days 26 13
14 Real World Turnaround Time for MTBC DST Specimen receipt to 1 st line DST by rapid broth: 4 to 6 weeks 2 nd line drugs: additional 2 to 4 weeks Referral to reference labs adds more time Molecular methods to detect resistance can help Molecular Detection of Drug Resistance Examining DNA of specific genes for mutations known to be associated with phenotypic resistance Rapid - analysis takes less than 1 day Can be done on culture isolates or directly on NAAT+ specimens 28 14
15 Sensitivity of Drug Resistance Mutations to Detect Drug Resistance Drug Gene % of Resist. Rifampin rpob 97% Isoniazid katg & inha 86% Ethambutol embb 79% Pyrazinamide pnca 86% Fluoroquinolones gyra 80% Injectables rrs 91% 29 CDC Molecular Detection of Drug Resistance (MDDR) Implemented Sept 2009 for isolates & expanded June 2012 for NAA+ specimens Test Indications Known/suspect DR case or contact to DR case Previous TB Treatment Patient from area with high rate of DR TB Large public health impact Mixed or nonviable culture 30 15
16 CDC Molecular Detection of Drug Resistance (MDDR) Provides 2-3 day DNA sequence analysis for drug resistance prediction 1 st and 2 nd line drugs tested MDDR complements conventional DST Used alone, MDDR and conventional DST are imperfect Used together, accuracy of drug resistance or susceptibility detection can be improved. Conventional DST results are still needed, or at least desirable, to confirm susceptibility to individual drugs. 31 Summary Powerful new rapid testing technologies allow laboratories to play a greater role in TB control Molecular tests offer rapid, reasonably accurate & predictive results that help fill in a medical & public health management gap that exists between specimen collection & the availability of conventional culture results Highly integrated systems-based approaches are essential to realize potential advantages from testing & information technologies 32 16
17 Thank you! 33 17
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