Diagnosis of TB: Laboratory Ken Jost Tuesday April 9, 2013
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1 TB Nurse Case Management San Antonio, Texas April 9-11, 2013 Diagnosis of TB: Laboratory Ken Jost Tuesday April 9, 2013 Ken Jost 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 21st Century Algorithm Process Specimen Amplificationbased Tests 24 hours AFB Smear Microscopy Inoculate Media 2-6 weeks Species Identification Molecular DST Drug Susceptibilities 2-3 weeks 3 Specimen Quality Accurate laboratory results are directly related to the quality of the specimen GOOD 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 and form - indicate test: initial Dx: NAAT isolation release: smear only drug resistance suspected? Transport to lab cool and quickly 4 2
3 Acid Fast Microscopy (AFB Smear) Rapid & universally available Used to support diagnosis and identify need to isolate Detects the most infectious cases Helps monitor response to therapy Identify priority cases for nucleic acid amplification (NAA) Not sensitive misses ~50% of TB Not specific in low TB prevalence areas (e.g. Texas) Positive smear may be NTM Highly specific where TB is highly prevalent 5 AFB Smear one microscopic field 6 3
4 TB Diagnostic Methods Related to # of Bacilli in Sputum Ref: Priorities for TB Bacteriology Services in Low-Income Countries, 2007, IUATLD 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 8 4
5 Nucleic Acid Amplification Tests (NAAT) Detects M. tuberculosis complex nucleic acids; does not distinguish between live and dead bacilli For initial Dx specimens only Not suitable for follow-up specimen or monitoring Sensitivity >95% for AFB smear-positive TB patients 55-75% of AFB smear-negative, culture-positive TB Does not replace culture 9 CDC Recommendations for NAAT MMWR, 2009, 58:7-10 NAAT should be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test result would alter case management or TB control activities NAAT now recommended as standard practice! 10 5
6 Who Should be Tested? CDC recommends NAAT on 1 st sputum of every TB SUSPECT for whom the test result would alter case management or TB control activities NAAT should NOT be ordered routinely if: Hospital/commercial lab already has NAAT+ Clin. Susp. is extremely high, e.g. pt. symptomatic, smear+, Dx=TB, on Rx i.e. when NAAT+ or result would not change actions Clin. Susp. very low, e.g. other Dx probable, spec is to r/o TB Definition of a TB suspect case can vary among providers TB programs, clinicians, and laboratorians must collaborate to develop criteria/definitions & policy for patients to be tested 11 How Do I Get a NAAT from the State Lab? DSHS automatically performs NAAT on smear positive respiratory specimens, effective 3/1/
7 Broth based system MGIT, Trek, MB/BacT AFB Culture Solid medium Purity Middlebrook agar & LJ 13 AFB Culture More sensitive than smear 5,000 to 10,000 AFB/ml for smear 10 to 100 AFB/ml for culture Required for drug susceptibilities & genotype Requires a quality specimen Positive for only ~85-90% of PTB Lengthy May be negative due to contamination 1-6 weeks by liquid media 2-8 weeks by solid media 14 7
8 Rapid Culture Identification DNA probes GenProbe HPLC (High performance liquid chromatography) Amplification-based tests Lab Developed Tests ( home brew ) Real time PCR Molecular Beacons DNA Sequencing Line Probes 15 How Do NAAT and Culture Compare? NAAT Culture Initial diagnosis Yes Yes Detect Non-viable Mtb Yes No Suitable to Monitor Treatment No Yes Detect Drug Resistance Some Yes Detect Drug Susceptible No Yes Genotype for Epidemiology No Yes Current NAATs are not sensitive enough to rule-out TB and they cannot replace culture 16 8
9 M. tuberculosis complex All positive by NAAT & AccuProbe Species No. of Texas strains * M. tuberculosis 8,058 (98.6%) M. bovis 79 (1.0%) M. bovis BCG 15 (0.2%) M. africanum 17 (0.2%) M. caprae M. microti M. canettii M. pinnipedii M TB Complex 17 * Data: Texas DSHS Laboratory Genotype Database Tuberculosis Genotyping What have been the most useful aspects of universal DNA fingerprinting of MTBC? Detecting previously unrecognized cases of transmission Reactivation vs. Reinfection ID of M. bovis & M. bovis BCG Detecting false positive cultures 18 9
10 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 19 M. tuberculosis cx Drug Susceptibility Testing 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 20 10
11 Drug Susceptibility Testing (DST) of Mycobacterium tuberculosis Complex Current Recommendations Initial isolate should be tested against primary or first-line drugs (FLD) INH, RMP, EMB, PZA For isolates resistant to RMP or to any 2 FLDs, test all second-line drugs To include FQ, AMK/KAN, CAP, ETH, PAS Not cycloserine; unreproducible 21 M. tuberculosis Drug Susceptibility Test Methods Growth Based Methods Broth Rapid (8-12 days) BACTEC MGIT 960 Agar Proportion Reference Method (21 days) Molecular Real time (almost) 11
12 Turnaround Time for MTBC Drug Susceptibility Testing (DST) Specimen receipt to 1 st line DST by rapid broth: 4 to 5 weeks 2 nd line drugs by rapid broth or agar proportion: additional 2 to 4 weeks Referral to reference lab adds more time Molecular methods can detect resistance to 1 st & 2 nd line drugs within 1 to 2 days Detection of Genetic Mutations Causing Resistance Examining DNA of specific genes for mutations known to be associated with conventional phenotypic resistance Rapid - analysis takes less 1 day Can be done on isolates or directly on NAA+ specimens! 24 12
13 MTBC Molecular Drug Resistance Testing Drug Gene % of Resist. Rifampin rpob 97% INH katg & inha 86% EMB embb 79% PZA pnca 86% F-quinolones gyra 80% Aminoglycosides rrs ~75% 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 Mixed or nonviable culture 26 13
14 CDC Molecular Detection of Drug Resistance (MDDR) Provides hr DNA sequence analysis for drug resistance prediction MDDR supplements, not replaces, conventional DST Used alone, MDDR and conventional DST are imperfect Used together, accuracy of the detection of drug resistance can be improved. Conventional DST results are still essential to confirm susceptibility to individual drugs. 27 Molecular Detection of Drug Resistance (MDDR) Service Offered by CDC MDDR Conventional DST Specimen Received PCR MGIT 960 DNA Sequencing Agar Proportion Molecular Results: 2 day TAT VS Conventional Results: 42 day TAT 28 14
15 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 29 Thank you! Acknowledgements Beverly Metchock Barbara Seaworth ken.jost@dshs.state.tx.us
16 Extra Y.O. Female Resident of Eagle Pass, TX on the Texas-Mexico border Day 0 (9/18/2012) sputum collected in Eagle Pass; patient critically ill Day 1 sputum received at DSHS-Austin Day 2 smear negative, GeneXpert Mtb detected, low, Rif R (Probe B) Day 2 sediment shipped to CDC for MDDR Day 6 Sanger: no amplification of RMP or INH targets Day 7 Pyrosequencing: no INH mutations, rpob mutation GAC>GTC; Asp516Val Predicts rifampin-r and rifabutin-s Day 11 patient started on Ethambutol, INH, Rifabutin, PZA, Levofloxacin, Linezolid, and Amikacin Day 21 MGIT 960 growth positive Day 22 Mtb cx ID d by HPLC Day 34 broth drug susceptibility: resistant to INH & rifampin; susceptible to rifabutin Day 44 agar proportion drug susceptibility: results confirmed broth results 32 16
17 Nucleic Acid Amplification Test Chart Nucleic Acid Amplification Tests Accuracy Tool - 6 Amplified Mycobacteria Laboratory tuberculosis (MTD ) Cepheid GeneXpert GenoType Developed Test AccuProbe Direct MTB/RIF MTBDRplus (LDT) Hain Company Gen-Probe, Inc Gen-Probe, Inc Cepheid n/a Nucleic Acid Yes Amplification? No Yes Yes Yes Identification MTBC from culture isolates Yes No No Not usually Sometimes Detection of MTBC in clinical specimens No Yes Yes Yes Yes Detection of mutations RIF, others associated with drug depending on resistance No No RIF RIF, INH platform Genes associated with rpo B, others depending on drug resistance detected none none rpo B kat G, inh A, rpo B platform Transcription Mediated Real time polymerase Amplification (TMA) to chain reaction (PCR) and Polymerase chain No amplifcation; amplify the ribosomal RNA molecular beacon reaction (PCR) to amplify Various methods DNA probe target followed by DNA technology to the genes followed by including PCR, real hybridizes to a probe hybridization to simultaneously amplify hybridization to specific time PCR, "molecular specific ribosomal detect the amplified and detect the rpob probes on nitrocellulose beacons," DNA Mechanism RNA target. target. gene. strips (line probe assay). sequencing. FDA approved Yes (cleared) Yes No No No Turnaround time from requires growth in specimen receipt in lab culture hrs hrs hrs hrs MTD, "Direct test," often the "Hain test" "Home brew," molecular beacons, Nick name probe, DNA probe mistakenly called "probe" GeneXpert, Xpert Line Probe Assay (LPA) PCR How reported -- Examples "Detected", "Positive," "Positive for M. "point mutation Positive result tuberculosis complex detected" Negative result "Not Detected" "no point mutation detected" RVCT no Agar Proportion Method Drug-free control quad: 90 colonies Isoniazid (INH) quad: 30 colonies Isoniazid 30/90 = 33% resistant Rifampin (R) quad: 23 colonies Rifampin 23/90 = 25% resistant Resistance = 1% or greater Curry Center Drug-Resistant Tuberculosis: A Survival Guide for Clinicians 34 17
18 Mutations Ability of mutations to predict resistance High confidence e.g. Ser531Leu Low confidence: limited literature reports Presence of mutation resistance Silent Neutral Increase MIC but < critical concentration Variable results Lack of mutation susceptible not all resistance mechanism known or targeted 35 How to Attain Turnaround Time Goals Systems Approach Pre- & Post- Analytical Efficient transport of specimens to and between labs Receive & Report electronically Analytical: Use rapid methods Concentrated fluorescence microscopy for AFB smears NAAT for Dx Liquid culture medium; Rapid ID/Rapid referral Refer primary culture If risk of drug resistance Molecular detection of drug resistance Concurrent 1 st and 2 nd line DST 36 18
19 CDC Report Index Case - Large N. Texas School Outbreak rifampin INH { ethambutol PZA quinolones injectables { 38 19
20 21st Century Algorithm Process Specimen Amplificationbased Tests 24 hours AFB Smear Microscopy Inoculate Media 2-6 weeks Species Identification Molecular DST Drug Susceptibilities 2-3 weeks 39 21st Century Algorithm Process Specimen Amplificationbased Tests 24 hours AFB Smear Microscopy Inoculate Media 2-6 weeks Species Identification Molecular DST Drug Susceptibilities 2-3 weeks 40 20
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