Research Methods for TB Diagnostics. Kathy DeRiemer, PhD, MPH University of California, Davis Shanghai, China: May 8, 2012
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1 Research Methods for TB Diagnostics Kathy DeRiemer, PhD, MPH University of California, Davis Shanghai, China: May 8, 2012
2 Overview Why do we need good TB diagnostics? What works? What doesn t work? How do we evaluate TB diagnostics? Can science inform policy?
3 Overview Why do we need good TB diagnostics? To find, treat and cure individuals To block transmission in the community To help monitor, evaluate and health programs and interventions, for example DOT, vaccine efficacy
4 Mycobacterium tuberculosis spreads through the air.... Coughing, sneezing, talking, shouting, singing...
5 Different stages of infection and disease M. tuberculosis Acute disease infection (30%) (10% life, HIV+ 5-10% /yr) Reactivation Exposure Latent (10% life, HIV+ 80%) infection Lifelong No M. tuberculosis containment infection (70%) (90%)
6 Spectrum of Disease Barry CE, et al. Nat Rev Microbiol 2009; 7,
7 Different Manifestations of Disease Pulmonary TB (~85% of all TB cases) Scrofula, TB in cervical lymph nodes Pott s disease, TB of the bone TB meningitis Ocular tuberculosis
8 Different Patient Populations
9 Challenges Which individuals are the most infectious? Which individuals cause the most disease in the community? How do we find these individuals who are superspeaders?
10 Overview Why do we need good TB diagnostics? What works? What doesn t work? How do we evaluate TB diagnostics? Can science inform policy?
11 Diagnosis of TB Infection (LTBI) and TB Disease Old tools still in use s Tuberculin skin test 1816 Stethoscope 1895 Chest radiographs sputum smears Culture BSL-3 lab Photo Credits: Francis J. Curry National Tuberculosis Center
12 Challenges Complexities of Mtb DNA extraction, amplification, and detection; biosafety concerns for manipulating M. tuberculosis Commercial nucleic acid amplification tests (NAAT) less sensitive than microbiological culture, especially for smear-negative TB Culture still necessary to extract DNA and for drug susceptibility tests Scale-up of labs and labs services slow and expensive Need major investments of infrastructure and human resources
13 Modern Technologies Line probe assays (LPAs) first significant breakthrough; detect RIF and INH resistance within 24 hour-48 hours Rapid, high-throughput technology to detect MDR-TB became available for reference lab level, sputmearpositive specimens only 2008 WHO endorses LPA, with guidance on several operational considerations for implementation LPAs still need sophisticated lab infrastructure and human resource skills WHO. Policy Statement: molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (MDR-TB)
14 Line Probe Assays Hain MDRTB/RIF First major breakthrough for TB diagnostics Detects RIF and INH resistance within hours Rapid, high throughput assay for smear-positive specimens Requires lab infrastructure and trained lab staff
15 Proposed evolutionary pathway of the tubercle bacilli Common ancestor of M. tuberculosis complex RD = region of difference, genomic deletion BCG Ernst JD, et al. J Clin Invest 2007; 117:
16 Maximum Parsimony Phylogeny of the M. tuberculosis complex M. tuberculosis complex MTBC Phylogeny derived using 89 concatenated gene sequences in 108 strains Hershberg R, et al. PLoS Biology 2008
17 Many tests, but.... Immunological tests Tuberculin skin test (TST) IFN-ᵞ release assays (IGRAs) Microscopy Ziehl Neelsen acid-fast stain; detect acid-fast bacilli (AFB+) Fluorochrome acid-fast stain Nucleic-Acid based tests NA amplification tests, PCR-based probes Culture Agar- or egg-based media Liquid broth media Identification Morphological characteristics and growth time Biochemical reactions Analysis of cell wall lipids, e.g. chromatography Nucleic acid probes Nucleic acid sequencing Diagnosis is still difficult!
18 What Do We Really Need? 1.Test with improved sensitivity 2. Test with minimal requirements for infrastructure, human resources 3. Test capable of providing rapid information on viability (for treatment programs) and drug resistance
19 Overview Why do we need good TB diagnostics? What works? What doesn t work? How do we evaluate TB diagnostics? Can science inform policy?
20 Characterize a Diagnostic Test Gold Gold Standard Disease No Disease Total Test result Positive a b a + b Negative c d c + d Total a + c b + d Positive Predictive Value (PPV) Negative Predictive Value (NPV) Sensitivity a/(a+c) Specificity d/(b+d)
21 Characterize a Diagnostic Test Gold Gold Standard Disease No Disease Total Test result Positive Negative Total Sensitivity = 370/490 = 75.5% PPV = 370/440 = 84.1% Specificity = 950/1020 = 93.1% NPV = 950/1070 = 88.8%
22 True positives (Sensitivity) Summary Receiver Operating Curve (SROC) AUC, Area under the curve False positive (1-Specificity)
23 Likelihood Ratios Uses all of the information from a test Likelihood ratio = P(Result Disease) P(Result No Disease) = Sensitivity / (1 Specificity) High likelihood ratio (>50): test result rules in a diagnosis Low likelihood ratio (close to 0): test result rules out disease Likelihood ratio = 1: test result provides no information at all about the likelihood of disease Prior odds of disease x likelihood ratio = Posterior odds
24 Is This Test Useful? Accurate? Sensitivity, specificity, PPV, NPV, SROCs, likelihood ratios Risks? Invasive procedure to get a specimen? Reproducible? Feasible? Does the test result have an effect on clinical decisions? Affects outcomes? Morbidity, mortality? Costs?
25 Overview Why do we need good TB diagnostics? What works? What doesn t work? How do we evaluate TB diagnostics? Can science inform policy?
26 Letter from India A Deadly Misdiagnosis Is it possible to save the millions of people who die from TB? by Michael Specter November 15, 2010
27 Interferon-γ Release Assays e.g. Quantiferon -TB-Gold in-tube assay ESAT-6, CFP-10
28 TB-Spot.TB Test ELISPOT assay Oxford Diagnostic Laboratories
29 Commercial Serological Antibody Detection Tests for the Diagnosis of Pulmonary Tuberculosis: A Systematic Review Steingart KR, et al. PLoS Med 2007; 4(6): e202
30 Many field studies were conducted! Sensitivity Specificity Steingart KR, et al. PLoS Med 2007; 4(6): e202
31 SROC Curves of Commercial Tests for Diagnosis of Pulmonary TB AUC = The curve is the regression line that summarizes the overall diagnostic accuracy
32 Smear microscopy-positive pulmonary TB AUC = Smear microscopy-negative pulmonary TB AUC =
33 None of the commercial tests evaluated perform well enough to replace sputum smear microscopy. Thus, these tests have little or no role in the diagnosis of pulmonary tuberculosis. Lack of methodological rigor in these studies was identified as a concern.... Steingart KR, et al. PLoS Med 2007; 4(6): e202. doi: /journal.pmed
34 WHO warns against the use of inaccurate blood tests for active tuberculosis A substandard test with unreliable results News release 20 July 2011 GENEVA - The use of currently available commercial blood (serological) tests to diagnose active tuberculosis (TB) often leads to misdiagnosis, mistreatment and potential harm to public health, says WHO in a policy recommendation issued today. WHO is urging countries to ban the inaccurate and unapproved blood tests and instead rely on accurate microbiological or molecular tests, as recommended by WHO.
35
36 GeneXpert System Photo: FIND
37 GeneXpert System GeneXpert platform, launched in 2004 Simplified molecular detection of anthrax bacilli Integrated and automated sample preparation, amplification and detection through PCR-based technology 6-color laser detection device, diagnosis within 100 minutes Can the Xpert system be adapted, for simultaneous detection of Mycobacterium tuberculosis and rifampin resistance? Took > 4 years to develop the Xpert MTB/RIF cartridge
38 GeneXpert System Unique collaboration between academia and industry, brokered by FIND (Foundation for Innovative New Diagnostics) Needs of users in the field dictated product specifications Academic partners collaborated on core components of the technology Adequate research funding from NIH and Bill & Melinda Gates Foundation Laboratory validation facilitated by existing resources, including FIND specimen bank
39 GeneXpert System Expression library representing all described rpob mutations enabled validation of rpob assay to detect rifampin resistance Libraries of other pathogens (mainly non-tuberculous mycobacteria) helped confirm specificity Large-scale field evaluations by FIND, using welldesigned operational research protocols Final product is a fully automated, closed (therefore safe) real-time PCR system, requiring only basic worker skills and no specialized laboratory structure
40 GeneXpert MTB/RIF test 2 hours from sample collection to test result! Boehme C, et al. N Engl J Med 2010; 363(11): doi: /NEJMoa
41 Performance in populations of 1000 individuals with varying TB prevalence WHO/HTM/TB/2011.2
42 Performance in populations of 1000 individuals with varying rifampin resistance WHO/HTM/TB/2011.2
43 WHO Policy Statement WHO HTM/TB/2011.4
44 WHO/HTM/TB/2011.2
45 Challenges of GeneXpert MTB/RIF System Needs stable electrical supply Needs temperature control (2⁰C 28⁰C kit storage, 30⁰C operating temperature) Maintenance off-site Expensive (>$17 per test) PPV of a single test indicating RIF resistance may be low RIF resistance is not a universally reliable proxy marker for MDR TB
46 Summary Why do we need good TB diagnostics? What works? What doesn t work? How do we evaluate TB diagnostics? Can science inform policy?
47 The Path Forward...
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