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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
GeneXpert for Detection of MTB and Rifampin
Resistance
David Warshauer, PhD, D(ABMM)
Deputy Director, Communicable DiseasesWisconsin State Laboratory of Hygiene
1
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
HEALTHYPEOPLE
20102020
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
14-14
Reduce TAT for laboratory Dx
Target: 2 days for 75%
[21 days // ’96]
U.S. Department of Health and Human Services, January 2000
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Percentage of Culture-Confirmed Pulmonary TB Cases Detected by NAAT
in Wisconsin (2005-2010)
2005 2006 2007 2008 2009 2010 (June)30
35
40
45
50
55
60
65
70
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
FDA-Cleared Molecular TB TestsFDA-Approved TB Molecular Assays
for Respiratory Specimens
Amplified M. tb Direct Test® (MTD): Gen-Probe, Inc.
Cepheid GeneXpert ®
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Nucleic Acid Amplification Tests
• Commercial tests available outside US
• BD ProbeTec™ MTB Direct Detection• COBAS® Amplicor® MTB Test• COBAS® TaqMan® MTB Test• Hain Genotype® Mycobacteria Series• Innogenetics INNO-LIPATM
• Laboratory Developed Tests• Off-label use of FDA-approved
tests
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
7
Courtesy Angela Starks, CDC
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Use of NAAT by US Public Health Laboratories in 2008 – Starks et al.
[CDC]
104,425 suspect TB patients
92,877 – not tested 88%
12,548 – tested 12%
NAR2010 – P 77
5,855 from Florida
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
GeneXpert System
Cepheid, Sunnyvale, CA
RT-PCR, < 2 hours
Potential for point of care testing
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
GeneXpert MTB/RIF Assay
• Automated commercial system for identification of M. tuberculosis complex and detection of rifampin resistance
• Decontamination, digestion, DNA extraction, amplification, and detection in same cartridge
• Integrated positive control assures that a negative result is not due to NAA inhibitors in the specimen
• Results in ~2 hours• Minimal hands on manipulation- technically simple• Platform is random access
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
GeneXpert
• Target: rpoB gene• Nested PCR and molecular beacon
technology• Same segment of the rpoB gene is used
for detection of both M. tb complex and rifampin resistance
• PCR amplifies a small region relevant for rifampin resistance; uses 5 probes to assess for mutations
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
12
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
GGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTG TCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTG
507
rpoB
533* ** * * *** **** * *
81 base pair core region* * *** ** ****
*** ****
InsertionTTC
InsertionTTCATG
DeletionCCATTC
DeletionGGCACC
DelAAC
DeletionCAGAAC
DeletionGACCAG
DeletionAATTCATGG
DeletionGAACAA
Genetics of Rifampin Resistance in M. tuberculosis
Adapted from Ramaswamy & Musser. 1998. Tubercule Lung Dis 79:3
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
M. Tb Complex PCR for All
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
finddiadnostics.org
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Example of Rif-Sensitive Profile – 5 probes are positive
MTB/Rif Assay design
Molecular Beacon
Target
Hybrid
Each probe is labeled with a different fluorophore, permitting simultaneous detection of the presence of wild type.
The MTB assay target is the 81 bp region (RRDR) of the rpoB gene.
SPC
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Challenges to Implementing NAAT Guidelines
• MTD: $30/test• Cepheid: $78/test (includes equipment & service [lease or cash]
& kit)
Gen-Probe® MTD Cepheid® MTB/RIF
Reagents2 controls3 patients $240 3 inhib ctrl
3 patients $234
Labor ($20/hr) 2 hrs $ 40 10 min $ 3
Direct costs per patient result
$ 93 $ 79
Courtesy Ken Jost, Texas SPHL
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Cepheid
• Limit of detection – 131 CFU/ml• M. tuberculosis viability – minus 8 log• 107 clinical specimens/suspicion of TB –
Vietnam– 100% - 29/29 AFB + / Culture +– 84.6% - 33/39AFB- /solid Culture +– 71.7% - 38/53AFB- / solid & broth Culture
Helb et al. JCM 48:229-237 (2010)
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
CepheidBoehme et al NEJM
• Assessed Xpert MTB/RIF in 1730 patients– Peru– Azerbaijan– South Africa– India
• Both suspected drug-sensitive and multidrug-resistant pulmonary TBBoehme, C.C. et al. NEJM 363: 1006-1015, 2010
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Boehme et al
• Among AFB smear pos/culture pos patients– Single Direct MTB/RIF identified 98.2%
(551/561)• Among AFB smear neg/culture pos
– 72.5% (124/171) – Addition of a second MTB/RIF increased sens to
85.1%– Addition of a third increased sens to 90.2%
• Specificity > 98.1%
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Rifampin ResistanceBoehme et al
• Detection of rifampin resistance
– Sensitivity of 99.1% (209/211)
– Specificity of 100% (506)
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Xpert Detection of Mtb in Pulmonary TB
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Rifampin Resistance Detection in Pulmonary TB
23
Chang, K. Journal of Infection (2012) pp 1-9
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Expert Performance in HIV Coinfected Population
24
Chang, K. Journal of Infection (2012) pp 1-9
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Xpert Performance Breakdown
25
Chang, K. Journal of Infection (2012) pp 1-9
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
2009 CDC Recommendations for use of NAAT
• “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 as standard practice
MMWR, 2009, 58:7-10
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
CDC Algorithm
• Collect at least one respiratory specimen, preferably the first, for NAAT
• Collect additional specimens for smear and culture
• Must interpret NAAT results in correlation with the AFB smear results
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
First Respiratory Specimen
Smear NegativeSmear Positive
NAATNAAT
Positive:Presumed TB,
Pending cultureresults
Negative
Use clinical judgment todetermine whether to begin therapy while
awaiting culture results and determine if
additional diagnostic testing is needed.
If a second specimen is smear positive,NAAT negative. the patient is presumed tohave an infection with non-tuberculousmycobacteria, pending culture results,
Consider testing another specimen (not to exceed a total of two).
NAAT Positive: A patient can bepresumed to have tuberculosis,pending culture results, if twospecimens are NAA positive.
Positive Negative
Consider testing another specimen (not to exceed a total of two).
Inhibitors Detected:Test result is of no
diagnostic help.Consider testing secondspecimen (not to exceed
a total of two).
Use clinical judgment todetermine whether to begin therapy while
awaiting culture results and determine if
additional diagnostic testing is needed.
Use clinical judgmentto determine whetherto begin therapy while
awaiting results ofculture and otherdiagnostic tests.
Currently availableNAA tests are not
sufficiently sensitiveto exclude the
diagnosis of TB inAFB smear negativepatients suspected
of having TB.
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Who should be tested?
• CDC recommends NAAT on first sputum of all patients SUSPECTED of TB for whom the test result would alter case management or TB control activities– NAAT should NOT be ordered routinely when
clinical suspicion of TB is low.
• Definition of a “suspect” case can vary among clinicians
• Clinicians, TB programs, and laboratorians must collaborate to develop criteria/definition for patients to be tested
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Wisconsin criteria for NAAT
• Signs and symptoms• Risk factors• Patient in airborne isolation • Reported to local health
department as a suspect case
• At WSLH all initial smear positive respiratory specimens automatically tested with NAAT
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
NAAT for release of patients suspected of pulmonary TB from
Isolation• CDC Expert panel
recommendations– Sputum that is NAAT negative and 2
additional sputums that are AFB smear negative.• Collected at 8-24 hour intervals, at least one
of which is an early morning specimen
– Should not be used when suspicion for TB is high enough to start TB medications.• Clinical response, usually 4-7 days treatment,
and 3 smear negative sputums
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Summary
• Advantages of NAAT– More rapid diagnosis– Initiation of earlier treatment– Cost savings with reduced patient
isolation– Faster reporting to TB Programs– Fewer transmissions
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
GeneXpert
• Will make NAAT more widely available to suspect TB patients– Earlier diagnosis– Approach 2020 goal
• Will provide rapid detection of rifampin resistance and possible MDR-TB cases– Caveat---In population with low
prevalence of rifampin resistance, predictive value will be poor (approx 56%) 33
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
GeneXpert
• NAAT is a supplemental test– Does not replace AFB smear and culture– Smear needed for interpretation– Culture still the “Gold Standard” for TB
diagnosis• In a low TB prevalence
population, most smear positive specimens will be NTMs
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Research Needs for Future Advancements
• Studies to develop, evaluate, and select the most effective and efficient NAAT and culture algorithms
• Develop better tests for non-respiratory specimens
• Develop tests with improved performance and ease-of-use
• Develop tests that will enhance the diagnosis of TB in children
• Develop multiplex assays that can detect M. avium complex, M. kansasii and other NTM
• Develop tests to detect resistance to both first and second line drugs
• Develop tests that can be used in resource limited countries. Requires ease of use and low cost.
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Thank You