1. Molecular testing for detection of Mycobacterium tuberculosis San Francisco Department of Public Health Laboratory

2. Aims
Molecular Testing for MTB: laboratory considerations and challenges
PCR-based testing at the SF Public Health Laboratory: GeneXpert (NOT FDA APPROVED)

3. Challenges for NAAT use

4. 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

5. 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

6. 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)

7. 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)

8. The San Francisco Public Health Lab Experience 2007 to present
PCR for TB
The San Francisco Public Health Lab Experience
2007 to present

9. 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

10. The Qiagen Real-Time: Light Cycler (uses an IS6110 target)
Sensitivity (culture as gold standard):
Smear many/numerous: 100%
Smear Few/Rare: %
Smear negative: %
Specificity : 97.6%
Data based on analysis of 108 prospective patient specimens
(sputum concentrates) and 50 frozen specimen

11. 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.

12. Switched to Cepheid, GeneXpert MTB/RIF -- NOT FDA APPROVED! --
2010
Switched to Cepheid, GeneXpert
MTB/RIF
-- NOT FDA APPROVED! --

13. The Device, Gene Xpert (Cepheid)
Single use cartridges
Extraction and amplification: in the cartridge
Fully Automated

14. Gene Xpert, results Using the Cepheid Gene Xpert: Clinical Specimen
Treat with NALC-
NaOH and make concentrate
Gene Xpert,
results

15. 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.

16. 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

17. Cepheid MTB: positive result
five probes
-assay has an
Internal PCR
Control
(for inhibition
assessment)
Test gives semi-quantitative results: “high”, “medium”, “low”, “very low” and “negative”

18. Cepheid MTB: negative result

19. Cepheid validation study

20. Validation: Sensitivity: sputum concentrates that became culture positive:
--13/13 smear numerous,
culture positives: %
--30/32 smear few / rare: %
(missed one “few” and one “rare”)
--29/40 smear negative,
culture positives: %
(Sm Neg/Cult Pos sensitivity of MTD test: ~72%
(according to package insert)

21. Specificity 30 negative sputum concentrates 0/30 positive Of those:
- 10 smear+ / culture positive MOTTs tested:
0 were reactive
--100% specificity

22. 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%

23. 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)

24. How we are using it How 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

25. Prospective results of GeneXpert Since April, 2010:
The two “misses” by GeneXpert were
each smear-negative.
All 15 “hits” were smear-positive.
(1 false call)

26. 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

27. 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