Xpert MTB/RIF March 2009. PAGE | 1 Agenda Background Current testings Xpert MTB/RIF product.
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Transcript of Xpert MTB/RIF March 2009. PAGE | 1 Agenda Background Current testings Xpert MTB/RIF product.
Xpert MTB/RIFMarch 2009
PAGE | 2
Agenda
• Background
• Current testings
• Xpert MTB/RIF product
Background
PAGE | 4
Slow Progress in Fight Against Tuberculosis
PAGE | 5
Why is Progress Slowing Down?
• Insensitive or too slow diagnostic methods => transmission
• Emergence of drug resistant strains, MDR and XDR
• “Only 2% of MDR-TB cases worldwide are being diagnosed and treated appropriately “ (WHO statement)
• Shortage of funding
Current tests
PAGE | 7
Common Laboratory Testing Algorithm
~ Week 2
~ Day 2
Day 1
Treat Sample Centrifuge RemoveSupernatent
Read SolidCulture
Liquid CultureResult Available
ReportPositive
SmearPositive?
PCRPositive?
Yes
Yes
No
No
PerformSensitivities
Prepare Smear Stain Smear Read Smear
Report SmearNegative
Report Smear PosPCR Negative
InoculateLiquid Culture
Inoculate SolidCulture
Report Liquid Culture
CulturePositive?
Yes
~ Week 3 - 4
Read SolidCulture
No
Re-incubate
ReadSensitivities
Report RifampicinResult
PerformSensitivities
CulturePositive?
Yes
Report Solid Culture
PAGE | 8
TB Smear Testing
• Time-to-result ~2 hrs
• Considerable hands-on-time
• Cheap
• Subjective
• Experienced technologists required
PAGE | 9
TB Smear Testing is Insensitive
• Sensitivity of smear testing is at most 70%. • 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 | 10
TB Culture Testing
• Time-to-result:• Solid culture: 6 weeks for a negative• Liquid culture: 3 weeks for a negative
• Possibility to grow other Mycobacterium species
• Experienced technologists required
PAGE | 11
Some Characteristics of an Ideal TB test
• 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
Xpert MTB/RIF product
PAGE | 13
Cartridge Design and Operating Principle
Sonicator Dome
Rotary Valve
Syringe Barrel RT-PCR
Tube
PAGE | 14
Bead Format Reagents
Target-specific reagent bead:
primers; rpoB specific probes; controls
Enzyme reagent bead:
Taq polymerase; dNTPs; Buffers; Mg 2+
Sample Preparation bead:
Bacillus globigii spores; excipients
Retaining balls
PAGE | 15
Intended use of Xpert MTB/RIF
• 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.
• The assay is intended for use on smear-positive and smear-negative samples.
• The assay, performed in the Cepheid GeneXpert® System*, is a semi-quantitative in-vitro diagnostic test
• 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 | 16
15 min incubation
Xpert MTB/RIF Protocol
STR
2 min hands-on
time to result = 1h45min
PAGE | 17
Molecular Beacon
Target
Hybrid
The Xpert MTB/RIF Molecular Beacon Assay
5 Probes bind to wild type
Probes do not bind to mutant sequence
1 Probe for SPC (B. globigii)
6 fluorescent dyes detected simultaneously
5’-3’-
-3’-5’
A
B
C D
E
rpoB gene
PAGE | 18
Sputum… the final PCR frontier
• It is usually highly viscous and thus incompatible with microfluidic devices
• It is often purulent
• It is often bloody
• Target organisms require concentration in order to be consistently detected
• Complicated off-line centrifugation and DNA extraction too slow and technically demanding for decentralized testing
PAGE | 19
The solution for sputum
Sample Treatment Reagent
•Liquidify sputum•Inactivates Mtb in sputum by 6 – 7 logs
PAGE | 20
Xpert MTB/RIF Preliminary Analytical Studies
• Analytical Sensitivity of approximately approx 100 cfu/ml (Smear 10,000 cfu/ml)
• Specificity tested with high concentrations of MOTT (Mycobacteria Other Than Tuberculosis)
• No evidence of amplicon cross-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
PAGE | 21
Preliminary Performance Characteristics
Sensitivity for Smear+/Culture+
>97%
Sensitivity for Smear-/Culture+
>90%
Overall Sensitivity >94%
Specificity for Smear-/Culture-
>95%
Sensitivity for Rifampicin Resistance
>95%
Sensitivity for Rifampicin Sensitivity
>95%
N.B. Data to be changed in final package insert
PAGE | 22
Semi-quantitative results in Xpert MTB/RIF
PAGE | 23
Amplification plot – TB positive/Rif sensitive
MTB Positive Medium, Rif Resistance NOT DETECTED
PAGE | 24
Amplification plots – TB positive/Rif resistance
MTB Positive Low, Rif Resistance DETECTED
PAGE | 25
Amplification plot – TB not detected
MTB not detected
PAGE | 26
Xpert MTB/RIF
• TB confirmation and therapeutic guidance in less than 2 hours
• More sensitive than smear, much quicker than culture
• Minimal technical expertise required
PAGE | 27
The test is:
• 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 pre-
treated 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