A rapid, fully-automated PCR system for sample processing diagnosis and rifampin

A rapid, fully-automated PCR system for
sample processing
processing, diagnosis
diagnosis, and rifampin
resistance detection of Mycobacterium
tuberculosis
Niklas Finnstrom, Martin Jones , Danica Helb, Ken Ho, JoAnn Kop, Peter Nguyen, Elizabeth
Story, Ellen Wallace, David Alland, Lee Christel, Michael Levi, David Persing, Richard
Rodgers,
g
, Hassan Safi,, Emily
y Winn-Deen,, Pete Dailey
y
Assay
y Design
g Goals





Sensitivity
S
iti it equall tto culture
lt
Rapid
Primary sample is raw sputum
very easy to use – minimal hands on effort
Sample treatment disinfects sputum for added
biosafety
 provide Rif resistance determination – surrogate
marker for MDR TB.
ugged, stable
stab e – refrigeration
e ge at o not
ot required.
equ ed
 rugged,
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rpoB Molecular Beacon Assay
rpoB 81 bp RRDR
5 Probes bind to wild type – produce signal
Probes do not bind to mutant sequence –
lack of signal
1 Probe for Sample Processing Control – B.
globigii target in spores
6 different fluorescent dyes detected
simultaneously
i lt
l iin a single
i l reaction
ti
Molecular
Beacon
Target
Hybrid
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Xpert MTB Protocol
Sample filtered and
washed in the GeneXpert
End of
hands on
work
Filter-captured organisms
ultrasonically lysed
to release their DNA
Disposable
Single Use Cartridge
DNA molecules mixed
with dry PCR reagents
Sample loaded into a cartridge
Approx 15 min
hands on work
Sputum liquefied with
Sample Treatment Reagent
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Mixture is delivered to
integrated reaction tube for
nested real-time
amplification and detection
Total time to result =
1h45min
GX-XVI system
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Xpert MTB Analytical Studies
 Analytical Sensitivity of approximately 50 - 100 cfu/ml
 Specificity tested with high concentrations of MOTT
 No evidence of amplicon cross
cross-contamination
contamination
 Perfect score on QCMD TB Proficiency Panel
 Mutation detection capability confirmed with isolate DNA
and artificial targets having a global frequency reported at
> 0.005
 Sample Treatment Reagent inactivates Mtb in sputum by
6 – 7 logs
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Xpert MTB Alpha Trial
Alpha Trial = test of development prototype
TRIAL SITES
Instituto de Medicina Tropicale “Alexander von Humboldt “
Universidad Peruana Cayetano Heredia (UPCH)
Bacteriological Laboratory
State Agency of Tuberculosis and Lung Diseases of Latvia
(SATLD)
Patients
P
ti t – individuals
i di id l suspected
t d off TB
Blinded study – Smear/culture and Xpert MTB
results determined by different groups
1 Patient result = combination of results from 3
p
1 GX only,
y, 1 culture only,
y, 1 split
p sample
p
samples:
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Alpha Trial TB Detection for Individual Patients
Per patient analysis for Mtb detection
TB CASE
DETECTION
TOTAL
95% CI
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Xpert
sensitivity
in smear+
smear+,
cul+
99.1%
99
1%
(113/114)
[95.2, 99.8]
Xpert
Xpert
sensitivity sensitivity
in smearsmear-,
in cul+
cul+
87.5%
(21/24)
[69.0,
95.7]]
97.1%
(134/138)
Xpert
specificity
in smearsmear-,
cul97.3%
97
3%
(219/225)
[94.3, 98.8]
Alpha
p Trial Rifampin
p Resistance Detection
Per
e pat
patient
e ta
analysis
a ys s for
o Rifampicin
a p c resistance
es sta ce
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RIF
RESISTANCE
DETECTION
Xpert
sensitivity in
Rif resistant
Xpert
specificity in
Rif sensitive
TOTAL
95% CI
100% (22/22)
[85 1 100]
[85.1,
100% (112/112)
[96 7 100]
[96.7,
Conclusions
 Rapid Mtb and Rif resistance detection
using sputum as the primary sample
 Excellent sensitivity and specificity – high
percentage of S
S-C+
C+ samples detected
 H
Hands-on
d
t k few
tasks
f
and
d simple:
i l add
dd STR tto
sample, shake to mix, add to cartridge, start
r n
run
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Acknowledgements
UMDNJ
Danica Helb
SATLD
Girts Skenders
Elizabeth Story
David Alland
UPCH
P
Pamela
l N
Nabeta
b t
FIND
Support: NIH grant: 52523 & The Foundation for Innovative Diagnostics
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