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Molecular testing for detection of
Mycobacterium tuberculosis
San Francisco Department of Public Health Laboratory
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Aims
• Molecular Testing for MTB: laboratory considerations and challenges
• PCR-based testing at the SF Public Health Laboratory: GeneXpert (NOT FDA APPROVED)
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Challenges for NAAT use
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2 FDAv approved NAA tests
• Roche PCR and Gen-Probe MTD test– the only FDA-approved options
– Roche test is leaving / has left
– MTD is expensive and time consuming
– MTD may be the most sensitive method
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Homebrew / RUO tests
• All are PCR -based
• Homebrew: rather inexpensive
• Performance can be excellent (See Halse & Musser JCM 2010, NYS lab)
But:
• They can require much more initial set-up / quality control
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NAAT methods: challenges
• Culture still rules: higher sensitivity
-higher specimen volume is tested by culture than by NAAT (MTD & GeneXpert may be exceptions)
-TB is a bit tougher than other organisms
-sputum often doesn’t contain many MTB organisms(compared to viral specimens (herpes, flu etc..) for example)
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Another challenge: logistics (# of specimens tested)
• Not necessarily a problem for all labs
• for medium, small labs: often very few specimens per day:
• Hence:
– you either run the entire assay on one or two specimens each day,
or
– you batch specimens, run one day a week, and lengthen your turn around times (and tick a lot of MD’s off)
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PCR for TB
The San Francisco Public Health Lab Experience
2007 to present
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Qiagen Real-Time(uses an IS6110 target)
• Automated extraction:Roche MagNAPure LC
• LightCycler real time PCR
• Internal inhibition control
• About 5 hours avg. from specimen to printed result
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The Qiagen Real-Time: Light Cycler (uses an IS6110 target)
Sensitivity (culture as gold standard):
• Smear many/numerous: 100%• Smear Few/Rare: 75%• Smear negative: 50%
Specificity : 97.6%
Data based on analysis of 108 prospective patient specimens (sputum concentrates) and 50 frozen specimen
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Qiagen RUO: issues
– Sensitivity was not as good as MTD
-we were getting 0 to 5 specimens per week, but running the test only on Wednesdays
-was costing a lot of micro time for that one day whether it was 1 or 5 specimens.
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2010
Switched to Cepheid, GeneXpertMTB/RIF
-- NOT FDA APPROVED! --
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The Device, Gene Xpert (Cepheid)
• Single use cartridges
• Extraction and amplification: in the cartridge
• Fully Automated
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Clinical Specimen
Gene Xpert,results
Using the Cepheid Gene Xpert:
Treat with NALC-NaOH and make concentrate
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Nested PCR: rpoB gene
• Take product of PCR 1, use as target in reaction 2
• Increase specificity by having two sets of primers needed for amplification
• Increase sensitivity by amplifying target prior to second PCR
1.
2.
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Target DNA sequence: rpoB gene
• The target of rifampin: RNA polymerase subunit B
• PCR amplifies a small region relevant for rifampin resistance; uses 5 probes to assess for mutations
probes
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Cepheid MTB: positive result
• five probes
-assay has anInternal PCRControl (for inhibitionassessment)
Test gives semi-quantitative results: “high”, “medium”, “low”, “very low” and “negative”
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Cepheid MTB: negative result
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Cepheid validation study
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Validation: Sensitivity:sputum concentrates that became culture
positive:
--13/13 smear numerous, culture positives: 100%
--30/32 smear few / rare: 94%(missed one “few” and one “rare”)
--29/40 smear negative, culture positives: 72.5%
(Sm Neg/Cult Pos sensitivity of MTD test: ~72% (according to package insert)
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Specificity
• 30 negative sputum concentrates– 0/30 positive
Of those:
- 10 smear+ / culture positive MOTTs tested:
0 were reactive
--100% specificity
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Summary of sensitivities described in the literature:
smear positive smear-negative
Moure et al (2011), JCM ND 75.30%
Boehme et al (2010) NEJM 98.20% 72.50%
Marlowe et al (2011) JCM 98% 72%
Helb et al (2010) JCM 98.40% 71.70%
Armand et al (2011) JCM 100% 48%
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Rif Resistance
• In search of specimens
• 3 MGIT samples of resistant isolates tested: all three were called correctly
• 3/85 (sputum) specimens were called Rif resistant– but phenotype testing showed otherwise
• (96.6% specific)
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How we are using itHow it is going…
• Sputum only (concentrated)
• Test is run: 3 days per week– Results follow: depending on when we get specimens:
either same day or next day
• All requests must go through public health TB control dept.
• No RIF susceptibility results reported right now: not validated
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Prospective results of GeneXpertSince April, 2010:
126 matched GeneXpert, culture,smear specimens
(since 4/2010)
17 confirmed MTB + :
15 positive by GeneXpert
2 negative by GeneXpert
11 positive for MOTT, neg. for MTB
11 negative byGeneXpert
109 negative byculture
109 negative byGeneXpert
3 called Rif resistantBy GeneXpert
2 Rif resistantby culture
(1 false call)
The two “misses” by GeneXpert wereeach smear-negative.
All 15 “hits” were smear-positive.
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Prospective results:
• 100% sensitivity for smear-positive
• 100% specificity
• 0/2 on smear negative specimens so far
• 67% PPV for rif resistance detection
• 100% NPV for rif resistance detection
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Acknowledgements
• Anna Babst, Senior Microbiologist• Jonathan Carlson, Microbiologist• Sally Liska, DrPH, Lab Director
• Masae Kawamura, MD TB Control• Houmpheng Banouvong TB Control• Luke Davis, MD, SFGH• Adithya Cattamanchi, MD, SFGH