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, GeneXpert
MTB/RIF
-- NOT FDA APPROVED! --
The Device, Gene Xpert (Cepheid)
• Single use cartridges
• Extraction and
amplification: in the
cartridge
• Fully Automated
Using the Cepheid Gene Xpert:
Clinical
Specimen
Treat with NALCNaOH and make concentrate
Gene Xpert,
results
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 an
Internal PCR
Control
(for inhibition
assessment)
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:
--30/32 smear few / rare:
100%
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
Moure et al (2011), JCM
Boehme et al (2010) NEJM
Marlowe et al (2011) JCM
Helb et al (2010) JCM
Armand et al (2011) JCM
ND
smear-negative
75.30%
98.20%
72.50%
98%
72%
98.40%
71.70%
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 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
Prospective results of GeneXpert
Since April, 2010:
126 matched
GeneXpert, culture,
smear specimens
(since 4/2010)
17 confirmed MTB + :
15 positive by
GeneXpert
2 negative by GeneXpert
3 called Rif resistant
By GeneXpert
2 Rif resistant
by culture
(1 false call)
11 positive for
MOTT, neg. for MTB
109 negative by
culture
11 negative by
GeneXpert
109 negative by
GeneXpert
The two “misses” by GeneXpert were
each smear-negative.
All 15 “hits” were smear-positive.
Prospective results:
• 100% sensitivity for smearpositive
• 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