TB Nurse Case Management. March 7-9, Diagnosis of TB: Ken Jost Wednesday March 7, 2012

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1 TB Nurse Case Management San Antonio, Texas March 7-9, 2012 Diagnosis of TB: Laboratory Ken Jost Wednesday March 7, 2012 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 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 Highly specific where TB is highly prevalent Not specific in low TB prevalence areas Positive smear may = NTM 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 AFB Culture More sensitive than smear 5,000 to 10, 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 May be negative due to contamination Lengthy 1-6 weeks by liquid media 2-8 weeks by solid media 8 4

5 Broth based system MGIT, Trek, MB/BacT AFB Culture Solid medium Purity Middlebrook agar & LJ Morphology - the Final Frontier! 9 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 10 5

6 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 MTB Complex 11 * Data: Texas DSHS Laboratory Genotype Database Mtb False Positive Cultures Burman & Reves, Clin Infect Dis 2000, 31: False positive are not rare Median false-positive rate = 31% 3.1% [range 2.2%-10.5%] 2% Clerical errors were as common as lab errors Single specimen positive was sensitive, but nonspecific indicator of false + Low colony count (solid medium) Thi k ibl Long time to positivity (broth medium) Think possible false + culture Contact lab and request genotype comparison 12 6

7 Tuberculosis Genotyping What have been the most useful aspects of universal DNA fingerprinting i of MTBC? Detecting false positive cultures ID of M. bovis & M. bovis BCG Uncovering previously unrecognized cases of transmission Reactivation vs. Reinfection 13 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 14 7

8 M. tuberculosis 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 15 M. tuberculosis Drug Susceptibility Test Methods Agar Proportion Method Reference Method (21 days) Broth-based Methods Rapid (8-12 days) MGIT 960, VersaTrek Molecular Real time (almost) 16 8

9 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 17 XDR-TB Extensively Drug Resistant Tuberculosis Isoniazid Ethambutol Rifampin control Streptomycin Ethionamide Ofloxacin control control Capreomycin Rifabutin Kanamycin 18 9

10 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 19 Nucleic Acid Amplification Tests (NAAT) Turnaround time measured in hours Detects M. tuberculosis complex nucleic acids; does not distinguish between live and dead bacilli Sensitivity >95% for AFB smear-positive TB patients 55-75% of AFB smear-negative, culture-positive TB Capability to detect drug resistance Does not replace culture 20 10

11 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 21 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 iti 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 22 11

12 Cepheid, Inc. GeneXpert MTB/Rif test 23 Boehme et al. NEJM 2010 Cepheid GeneXpert MTB/Rif Practical NAAT 15 minute entry-level technician vs. 3-4 hrs highly skilled technician Highly accurate for smear positive TB Sensitivity uncertain for smear negative TB Limited data Rifampin-R Extra-pulmonary (CSF, gastric..) 24 12

13 Xpert Mtb/Rif Performance Mtb detection Year Total Author pub. Sites specimens Sensitivity Specificity smr+ cult+ smr- cult+ Boehme global 1, /561 (98%) 124/171 (72%) 604/609 (99%) Marlowe U.S /87 (97%) 31/43 (72%) 83/87 (95%) Theron S. African /94 (95%) 12/22 (55%) 320/339 (94%) Accuracy for Mtb ID comparable to other NAATs Detects likely TB cases missed by culture Accurately R/U rifampin resistance 25 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 26 13

14 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 28 14

15 MTBC Molecular Drug Resistance Testing Drug Gene % of Resist. RMP INH INH-ETH EMB PZA F-quinolones rpob ~96% katg 40-60% inha 15-43% embb ~60% pnca ~75% gyra ~75% Aminoglycosides rrs ~75% Modified from Curry Center: Drug-resistant tuberculosis A survival guide for clinicians, 2nd Edition Mutations Ability of mutations to predict resistance High confidence e.g. Ser531Leu Low confidence: limited 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 30 15

16 CDC Molecular Detection of Drug Resistance (MDDR) Implemented Sept 2009 Provides 48 hr DNA sequence analysis for drug resistance prediction CDC Dear Colleagues letter Feb 2011 MDDR supplements, not replaces, conventional DST Testing and interpretation coordinated through RTMCC/TB Program & state PHL 31 CDC MDDR 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 Sample : culture positive isolate usually Smear positive specimen on occasion Reports: CDC faxes to 1 lab & 1 program contact Program contact assures results are shared with local/regional/state providers 32 16

17 HeartLand Texas DSHS Lab Coordination HeartLand Consulted on patient w/ drug resistance risks Notifies DSHS-Austin & together coordinate locating isolate for CDC test & performing 2 nd line drugs DSHS-Austin Lab Notifies HeartLand when: DST shows early signs of drug resistance Patient is persistent culture positive; 2 nd DST performed 33 Early Detection of Drug Resistant TB: Public Health Emergency 7/26/11: 39 yo high school teacher with 10 mos cough, recent fever, sweats; lived in South Africa for 10 years Cavitary changes on chest CT, sputum and BAL AFB smear (+), NAAT (+) MTB 8/3/11 presented with pneumothorax and cavitary densitites on CXR Started on IREZ 17

18 Public Health Lab Response Index Case Large N. Texas School Outbreak 35 Early Detection of Drug Resistant TB: Public Health Emergency No MTB mutations associated with drug resistance Culture grows M TB sensitive to all 1 st line drugs 18

19 Early Detection of Drug Resistant TB: Public Health Emergency Contact investigation started in school involving i hundreds d of students, t teachers and administrators Contact investigation yields: ~ 13 active TB cases ~ 250 LTBI cases TB cases started on IREZ LTBI cases started (mostly) on INH The ripples are still being felt Summary Powerful new rapid testing technologies allow laboratories to play a greater role in TB control Rapid methods are adjunctive & costly, but not as costly as delay in diagnosis and treatment Highly integrated systems-based approaches are essential to realize potential advantages from testing & information technologies 38 19

20 Thank you Acknowledgements Beverly Metchock David Warshauer Barbara Seaworth David Griffith

21 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 41 DSHS Rifampin Conventional DST vs. CDC rpob Mutations Nov 2009 Nov 2011 Conventional DST rpob mutation RMP-R RMP-S total yes 29* 1 30 no total * 4 isolates were agar-r and Bactec-S sensitivity 97% specificity 97% PPV 97% NPV 97% prevalence 47% 42 21

22 DSHS Isoniazid Conventional DST vs. CDC katg & inha Mutations Nov 2009 Nov 2011 Conventional DST mutation INH-R INH-S total yes no total sensitivity 88% specificity 100% PPV 100% NPV 81% prevalence 64% 43 CDC Report Index Case - Large N. Texas School Outbreak rifampin INH { ethambutol PZA quinolones injectables { 44 22

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