Molecular testing for detection of Mycobacterium tuberculosis San Francisco Department of Public Health Laboratory
Mar 31, 2015
Molecular testing for detection of
Mycobacterium tuberculosis
San Francisco Department of Public Health Laboratory
Aims
• Molecular Testing for MTB: laboratory considerations and challenges
• PCR-based testing at the SF Public Health Laboratory: GeneXpert (NOT FDA APPROVED)
Challenges for NAAT use
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
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
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)
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)
PCR for TB
The San Francisco Public Health Lab Experience
2007 to present
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
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
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.
2010
Switched to Cepheid, GeneXpertMTB/RIF
-- NOT FDA APPROVED! --
The Device, Gene Xpert (Cepheid)
• Single use cartridges
• Extraction and amplification: in the cartridge
• Fully Automated
Clinical Specimen
Gene Xpert,results
Using the Cepheid Gene Xpert:
Treat with NALC-NaOH and make concentrate
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.
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
Cepheid MTB: positive result
• five probes
-assay has anInternal PCRControl (for inhibitionassessment)
Test gives semi-quantitative results: “high”, “medium”, “low”, “very low” and “negative”
Cepheid MTB: negative result
Cepheid validation study
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)
Specificity
• 30 negative sputum concentrates– 0/30 positive
Of those:
- 10 smear+ / culture positive MOTTs tested:
0 were reactive
--100% specificity
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%
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)
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
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.
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
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