Transcript GeneXpert - Wisconsin State Laboratory of Hygiene

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GeneXpert for Detection of
MTB and Rifampin Resistance
David Warshauer, PhD, D(ABMM)
Deputy Director, Communicable Diseases
Wisconsin State Laboratory of Hygiene
WISCONSIN STATE
LABORATORY OF HYGIENE
HEALTHY
PEOPLE
2010
2020
WISCONSIN STATE
LABORATORY OF HYGIENE
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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 HYGIENE
Percentage of Culture-Confirmed Pulmonary
TB Cases Detected by NAAT in Wisconsin
(2005-2010)
70
65
60
55
50
45
40
35
30
2005
WISCONSIN STATE
LABORATORY OF HYGIENE
2006
2007
2008
2009
2010 (June)
FDA-Cleared Molecular TB Tests
FDA-Approved TB Molecular Assays
for Respiratory Specimens
Amplified M. tb Direct Test®
(MTD): Gen-Probe, Inc.
Cepheid GeneXpert ®
WISCONSIN STATE
LABORATORY OF HYGIENE
Nucleic Acid Amplification Tests
• Commercial tests available outside US
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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 HYGIENE
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Courtesy Angela Starks, CDC
WISCONSIN 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%
5,855 from Florida
WISCONSIN STATE
LABORATORY OF HYGIENE
NAR2010 – P 77
GeneXpert System
Cepheid, Sunnyvale, CA
RT-PCR, < 2 hours
Potential for point of care testing
WISCONSIN STATE
LABORATORY OF HYGIENE
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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
WISCONSIN 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 HYGIENE
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LABORATORY OF HYGIENE
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Genetics of Rifampin Resistance in M.
tuberculosis
rpoB
Deletion
Deletion
Deletion
AATTCATGG GACCAG GAACAA
Deletion
CCATTC
Deletion
CAGAAC
Insertion Insertion
TTCATG
TTC
Deletion
GGCACC
Del
AAC
GGCACCAGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTG TCGGGGTTGACCCACAAGCGCCGACTGTCGGCGCTG
507
* ********** * *** *
* *
81 base pair core region
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LABORATORY OF HYGIENE
******
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* 533
Adapted from Ramaswamy & Musser. 1998. Tubercule Lung Dis 79:3
M. Tb Complex PCR for All
WISCONSIN STATE
LABORATORY OF HYGIENE
finddiadnostics.org
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LABORATORY OF HYGIENE
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MTB/Rif Assay design
The MTB assay target is the 81 bp region (RRDR) of the rpoB gene.
Molecular
Beacon
Target
Hybrid
Each probe is labeled with a different fluorophore,
permitting simultaneous detection of the presence
of wild type.
WISCONSIN STATE
LABORATORY OF HYGIENE
SPC
Example of Rif-Sensitive Profile – 5 probes are
positive
Challenges to Implementing NAAT
Guidelines
Gen-Probe® MTD
Cepheid® MTB/RIF
Reagents
2 controls
3 patients $240
3 inhib ctrl
3 patients
$234
Labor
($20/hr)
2 hrs
10 min
$ 3
Direct costs
per patient
result
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•
$ 40
$ 93
$ 79
Courtesy Ken Jost, Texas SPHL
MTD: $30/test
Cepheid: $78/test (includes equipment & service [lease or cash] & kit)
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LABORATORY OF HYGIENE
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Cepheid
• Limit of detection – 131 CFU/ml
• M. tuberculosis viability – minus 8 log
• 107 clinical specimens/suspicion of TB –
Vietnam
– 100% - 29/29
– 84.6% - 33/39
– 71.7% - 38/53
AFB + / Culture +
AFB- /solid Culture +
AFB- / solid & broth Culture
Helb et al. JCM 48:229-237 (2010)
WISCONSIN STATE
LABORATORY OF HYGIENE
Cepheid
Boehme et al NEJM
• Assessed Xpert MTB/RIF in 1730
patients
– Peru
– Azerbaijan
– South Africa
– India
• Both suspected drug-sensitive and
multidrug-resistant pulmonary TB
Boehme, C.C. et al. NEJM 363: 1006-1015, 2010
WISCONSIN 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 HYGIENE
Rifampin Resistance
Boehme et al
• Detection of rifampin resistance
– Sensitivity of 99.1% (209/211)
– Specificity of 100% (506)
WISCONSIN STATE
LABORATORY OF HYGIENE
Xpert Detection of Mtb in Pulmonary
TB
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Rifampin Resistance Detection in
Pulmonary TB
Chang, K. Journal of Infection (2012) pp 1-9
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LABORATORY OF HYGIENE
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Expert Performance in HIV
Coinfected Population
Chang, K. Journal of Infection (2012) pp 1-9
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Xpert Performance Breakdown
Chang, K. Journal of Infection (2012) pp 1-9
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
MMWR, 2009, 58:7-10
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 HYGIENE
First Respiratory Specimen
Smear Positive
Smear Negative
NAAT
Positive:
Presumed TB,
Pending culture
results
Negative
Use clinical judgment to
determine whether to
begin therapy while
awaiting culture results
and determine if
additional diagnostic
testing is needed.
NAAT
Inhibitors Detected:
Test result is of no
diagnostic help.
Consider testing second
specimen (not to exceed
a total of two).
Consider testing another specimen
(not to exceed a total of two).
If a second specimen is smear positive,
NAAT negative. the patient is presumed to
have an infection with non-tuberculous
WISCONSIN
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mycobacteria,
pending culture results,
LABORATORY OF HYGIENE
Positive
Use clinical judgment to
determine whether to
begin therapy while
awaiting culture results
and determine if
additional diagnostic
testing is needed.
Consider testing another specimen
(not to exceed a total of two).
NAAT Positive: A patient can be
presumed to have tuberculosis,
pending culture results, if two
specimens are NAA positive.
Negative
Use clinical judgment
to determine whether
to begin therapy while
awaiting results of
culture and other
diagnostic tests.
Currently available
NAA tests are not
sufficiently sensitive
to exclude the
diagnosis of TB in
AFB smear negative
patients suspected
of having TB.
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 HYGIENE
Wisconsin criteria for NAAT
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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
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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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LABORATORY OF HYGIENE
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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 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%)
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LABORATORY OF HYGIENE
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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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LABORATORY OF HYGIENE
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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 HYGIENE
Thank You
WISCONSIN STATE
LABORATORY OF HYGIENE