Xpert MTB/RIF
March 2009
Agenda
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Background
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Current testings
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Xpert MTB/RIF product
PAGE | 1
Background
Slow Progress in Fight Against Tuberculosis
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Why is Progress Slowing Down?
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Insensitive or too slow diagnostic methods => transmission
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Emergence of drug resistant strains, MDR and XDR
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“Only 2% of MDR-TB cases worldwide are being diagnosed and
treated appropriately “ (WHO statement)
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Shortage of funding
PAGE | 4
Current tests
Common Laboratory Testing Algorithm
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TB Smear Testing
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Time-to-result ~2 hrs
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Considerable hands-on-time
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Cheap
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Subjective
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Experienced technologists required
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TB Smear Testing is Insensitive
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Sensitivity of smear testing is at most 70%.
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A minimum of 30% are smear-negative culture-positive1
Approximately 20% of transmitted infections are caused by
smear-negative / culture positive patients 2
Sources:
1:Long R, Smear-Negative Pulmonary Tuberculosis in Industrialized Countries, Chest 2001;120;330-334
2:Behr MA, Warren SA, Salamon H, et al. Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli,
Lancet 1999; 353:444–449
PAGE | 8
TB Culture Testing
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Time-to-result:
Solid culture: 6 weeks for a negative
• Liquid culture: 3 weeks for a negative
Possibility to grow other Mycobacterium
species
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PAGE | 9
Experienced technologists required
Some Characteristics of an Ideal TB test
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Direct-specimen detection of
MTB
Sensitivity of culture with goal of
eliminating negative cultures
Simultaneous detection of drug
resistance
On demand availability (no
batching requirement)
Decentralized platform
technology to reduce or eliminate
sample shipment
Rapid (<2 hours)
Portable
Low skill requirement
Highly reliable
PAGE | 10
Xpert MTB/RIF product
Cartridge Design and Operating Principle
Syringe
Barrel
RT-PCR
Tube
Rotary
Valve
Sonicator
Dome
PAGE | 12
Bead Format Reagents
Retaining
balls
Sample Preparation bead:
Bacillus globigii spores; excipients
Enzyme reagent bead:
Taq polymerase; dNTPs; Buffers; Mg 2+
Target-specific reagent bead:
primers; rpoB specific probes; controls
PAGE | 13
Intended use of Xpert MTB/RIF
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The Xpert MTB/RIF Assay is intended for rapid detection of
Mycobacterium tuberculosis complex and rifampicin resistance in
adult patients in sputum specimens with clinical suspicion of
tuberculosis.
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The assay is intended for use on smear-positive and smear-negative
samples.
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The assay, performed in the Cepheid GeneXpert® System*, is a semiquantitative in-vitro diagnostic test
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The Xpert MTB/RIF assay is not intended for use in
treatment monitoring.
* N.B. Xpert MTB runs on GeneXpert system 6 colour with software version 2.1. only!
PAGE | 14
Xpert MTB/RIF Protocol
2 min hands-on
15 min incubation
STR
time to result = 1h45min
PAGE | 15
The Xpert MTB/RIF Molecular Beacon Assay
C
A
5’3’-
D
-3’
-5’
rpoB gene
E
B
5 Probes bind to wild type
Probes do not bind to mutant sequence
1 Probe for SPC (B. globigii)
Molecular
Beacon
6 fluorescent dyes detected simultaneously
Target
Hybrid
PAGE | 16
Sputum… the final PCR frontier
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It is usually highly viscous and thus incompatible with microfluidic
devices
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It is often purulent
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It is often bloody
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Target organisms require concentration in order to be
consistently detected
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Complicated off-line centrifugation and DNA extraction too slow
and technically demanding for decentralized testing
PAGE | 17
The solution for sputum
Sample Treatment Reagent
•Liquidify
sputum
•Inactivates Mtb in sputum by 6 – 7 logs
PAGE | 18
Xpert MTB/RIF Preliminary Analytical Studies
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Analytical Sensitivity of approximately approx 100 cfu/ml (Smear 10,000
cfu/ml)
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Specificity tested with high concentrations of MOTT (Mycobacteria Other
Than Tuberculosis)
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No evidence of amplicon cross-contamination
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Perfect score on QCMD TB Proficiency Panel
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Mutation detection capability confirmed with isolate DNA and artificial targets
having a global frequency reported at > 0.005
PAGE | 19
Preliminary Performance Characteristics
Sensitivity for
Smear+/Culture+
Sensitivity for
Smear-/Culture+
Overall Sensitivity
Specificity for
Smear-/Culture-
>90%
>94%
>95%
Sensitivity for
Rifampicin Resistance
>95%
Sensitivity for
Rifampicin Sensitivity
>95%
N.B. Data to be changed in final package insert
PAGE | 20
>97%
Semi-quantitative results in Xpert MTB/RIF
PAGE | 21
Amplification plot – TB positive/Rif sensitive
MTB Positive Medium, Rif Resistance NOT DETECTED
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Amplification plots – TB positive/Rif resistance
MTB Positive Low, Rif Resistance DETECTED
PAGE | 23
Amplification plot – TB not detected
MTB not detected
PAGE | 24
Xpert MTB/RIF
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TB confirmation and therapeutic guidance in less than
2 hours
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More sensitive than smear, much quicker than culture
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Minimal technical expertise required
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The test is:
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PAGE | 26
Available as from 2nd Quarter 2009
Rapid, with results delivered in less than two hours
High in sensitivity and specificity, for smear-positive
and smear-negative samples
A walk-away ease of use system: just insert pretreated samples
A closed cartridge; sample preparation,
amplification and detection take place in the
cartridge
Detecting M tuberculosis and rifampicin resistance
utilizing the rpoB gene
Acknowledgements
UMDNJ, New Jersey, USA
The Foundation for Innovative Diagnostics (FIND),
Geneva, Switzerland
Support:
NIH grant: 52523
The Foundation for Innovative Diagnostics
Xpert MTB/RIF CE-IVD (10)
Product code: GXMTB-10