Xpert MTB/RIFMarch 2009

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Agenda

• Background

• Current testings

• Xpert MTB/RIF product

Background

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Slow Progress in Fight Against Tuberculosis

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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

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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

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TB Smear Testing

• Time-to-result ~2 hrs

• Considerable hands-on-time

• Cheap

• Subjective

• Experienced technologists required

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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

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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

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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

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Cartridge Design and Operating Principle

Sonicator Dome

Rotary Valve

Syringe Barrel RT-PCR

Tube

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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

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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!

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15 min incubation

Xpert MTB/RIF Protocol

STR

2 min hands-on

time to result = 1h45min

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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

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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

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The solution for sputum

Sample Treatment Reagent

•Liquidify sputum•Inactivates Mtb in sputum by 6 – 7 logs

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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

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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

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Semi-quantitative results in Xpert MTB/RIF

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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

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Amplification plot – TB not detected

MTB not detected

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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  

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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