Rapid Diagnostic Techniques for Identifying Tuberculosis Ken Jost November 13, 2008
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1 Tuberculosis Updates for Clinicians San Antonio, Texas November 13, 2008 Rapid Diagnostic Techniques for Identifying Tuberculosis Ken Jost November 13, 2008 Rapid Diagnostic Techniques for Identifying Tuberculosis Ken Jost Mycobacteriology Laboratory Texas Department D of State HealtH ealth Services Heartland TB Updates for the Clinician November 13,
2 3 4 2
3 TB Laboratory Diagnostic Services 19 th th /20 th th Century specimen smear culture identification drug susceptibility 5 TB Laboratory Diagnostic Services 21 st st Century specimen smear if diagnostic specimen rapid culture direct identification (NAAT +) rapid identification & dna fingerprint molecular MDR DST rapid drug susceptibility 1 st & 2 nd line 6 3
4 TB is considered a clinically ically based diagnosis, but some clinicians delay treatment until laboratory results are available Test results must be available ASAP to reduce delay in initiation of therapy Pascopella la et al.,, 2004, J. Clin. Microbiol. 42:4 : Laboratory Reporting of Tuberculosis Test Results & Patient Treatment Initiation AFB smea ear- positive patients AFB smea ear- negative patients N Reporting timeframe median [range] Treatment init itiation median [range] [0-35] 1.0 [0-70] [9 143] 22.0 [0 145] Ref: Pascopella et al., 2004, JCM 42:
5 Recommended TB Turnaround Times 0 Receive specimen, process 1 2 NAAT Molecular MDR DST 21 1 st line Drug Susceptibilities* (2 nd line DST: 4 wks after test order) Days Smear For 80% o ff dii ag nosti ic specimens i Culture Identification* Report AFB culture negative 9 Diagnostic Methods for TB Related to # of Bacilli Present in Sputum Ref: Priorities for TB Bacteriology Services in Low -Income Countries, 2007, IUATLD 10 5
6 Nucleic Acid Amplification Tests (NAAT) How well does NAAT perform? Who should be tested? 11 FDA approvals for NAATs MTD AFB+ Sputum Dec 95 AMPLICOR Roche for AFB+ Sputum 1996 Enhanced MTD AFB- Sputum 1999 AMPLICOR COBAS 1999 MMWR September 10, 1993 Not FDA-approved November 1, 1996 Interim guidelines July 7, 2000 Update on NAATs? Being updated 12 6
7 NAAT Performance 13 Reduction in turnaround time for laboratory diagnosis of pulmonary TB by routine use of NAAT Processing: 5 days; NAAT 4 days; broth medium monitored 7 days NAAT (first specimen) AFB and culture (3 specimens) 797 pt [81 TB] Assay Sens Spec PPV NPV Mean TAT AFB NAAT Cult Moore et al., 2005, DMID 52:
8 Current CDC Recommendations for NAAT Collect sputum on 3 different days Perform NAAT on the 1 st specimen, 1 st smear+ specimen, & additional as indicated Number of specimens to test? Clinical situation Prevalence of o TB Prevalence of o NTM Laboratory proficiency MMWR, MMWR, 2000, 2000, 49:593 49: CDC Algorithm If NAAT+ & smear+, presume e TB, no additional NAAT If NAAT+ & smear- & test 2 nd specimen If NAAT +, presume TB If NAAT-,, test 2 nd specimen and for inhibitors If inhibitor+,, NAAT is of no help If NAAT-,, presume p not infectious (if smear neg) 16 8
9 Who Should be Tested? CDC recommends NAAT for all patients suspected of TB Others suggest not testing patients ts with low suspicion of TB Others suggest not using NAAT when very high clinical suspicion Several investigators suggest to use NAAT AT to confirm TB in patients with medium-high likelihood of disease NAAT+ AT+ especially valuable in smear- and extrapulmonary 17 Every test performed without a strict indication, is not only spinning the wheels, it also means that the quality of a more meaningful test is jeopardized. Over-use of a test Under-use Mis-use Cou Cou rtesy rtesy Max Max Salfinger Sal finger = ERRORs Co-Discoverer of Interferon 18 9
10 Tuberculosis Genotyping What have been the most useful aspects of universal DNA fingerprinting of TBC? Detecting false positive cultures Uncovering previously unrecognized cases of transmission Rapid ID of M. bovis (& BCG) Assessing efficacy of TB control programs 19 Mtb Drug Susceptibility Test Methods Broth-based Methods Rapid Agar Proportion Method Confirmatory Molecular Real time (almost) 20 10
11 Standardized recommended test concentrations Drug 7H10 agar Bactec 460 MGIT 960 Rifampin INH 0.2 / / / 0.4 Ofloxacin 2 2? Moxifloxacin??? Capreomycin ? Kanamycin 5 5? Amikacin 4 1? 21 Molecular DST Methods Sequencing Line Probe Assays Hain Innogenetics Molecular Beacons Microarrays 22 11
12 Molecular Mechanisms of Resistance Drug RIF INH PZA EMB FQ KAN AMK CAP Gene % rpob >95 inha, katg, ndh, ahpc 80 pnca embb, embc, embr gyra, gyrb rrs 75 rrs >75 rrs, tyla ND 23 Hain MTBDRplus Procedure 24 12
13 25 Hain MTBDRplus 536 Smear-positive Patients, Cape Town, S. Africa Am Am J J Respir RespirCrit Crit Care Care Med, Med, 2008, 2008, 177: :
14 Molecular Beacons aka R eal-time PCR PCR and detection take place in single tube Turnaround time about 4 hours Available for Rif & INH Accuracy so far > 96% 27 Molecular Beacons for Detecting Rifampin Resistance 3 year s of of CA CA expe erience with cultures and sp sp ecimen sediments Molecular Culture rifampin Culture rifampin beacon result resistant susceptible Mutation 39 5 detected Mutation not detected Beacons test 2 5 inconclusive Prevalence =16%, # of of samples s =260; (97% %) ) eval eval uable, se se nsitivity & specific s ity ity = 97% 97% 28 14
15 29 DNA Microarray Walk away automation High throughput Many targets tested 30 15
16 Problems with Molecular DST? Not practical for all drugs Many reactions necessary Not alla mechani anisms of resistance known; Not all resista istance detected by mutations Not all mutations = resistance strains may have a mutation consc onsistent with resistancer and a DST result interpreted as susceptible. Discorda ordance between methods Emerging resistance in mixed populations may not be detected Helpful when you detect a mutation; not so s much when you do not (cannot r/o R) 31 Limitations of Molecular DST Validated for cultures and smr+ + sputum specimens only (not( smr- & extra-pulmonary) Sensitivity for INH-R R only ~80%, worse for EMB Molecular methods can be used to guide treatment until conventional DST results are available (usually about a month later) 32 16
17 M48-A Safety Levels of lab and referral services Clinical significance of Mycobacterium spp. Specimen types, t collection, transport and storage Detection and ID methods 33 How to Attain Turnaround Time Goals Pre- & Post- Analytical Overnight deli livery of spec ecimens Report & Receive electroni tronically Know your customer ers! Systems Approach Analytical: Use rapid d methods Concentrated ed fluores orescence microscopy for acida fast smears ears Include e liquid culture ure medium dium rapid identificati entification methods Molecul cular r MDR [TB Program/doc approval] al] Report and a d call critical positive results s the same day! Don t t waitw it: : Communicat cate e and Anticip ticipate MDR & Concurrent second-li -line e DST Referrals 34 17
18 Laboratory Challenges Efficient transport of specimens to the laboratory or between network laboratories Interaction among public health & clinical labs & treating physicians & TB Controllers Lack of standard test algorithms for rapid technology use Maintaining staff proficiency Antiquated lab information systems; fully integrated lab information management and reporting systems 35 Summary Rapid testing technology allow labs 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 36 18
19 Acknowledgements Max Salfinger Dave Warshauer Bev Metchock Ed Desmond 37 TB Control NAAT MDR Kennedy Space Center, Florida 38 19
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