Xpert MTB/RIF March 2009

Agenda Background Current testings Xpert MTB/RIF product PAGE | 1

Background

Slow Progress in Fight Against Tuberculosis PAGE | 3

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 PAGE | 4

Current tests

Common Laboratory Testing Algorithm PAGE | 6

TB Smear Testing Time-to-result ~2 hrs Considerable hands-on-time Cheap Subjective Experienced technologists required PAGE | 7

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 | 8

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 | 9

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 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 Sample Preparation bead: Enzyme reagent bead: 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 13

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 | 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 5’- 3’- -3’ -5’ A B C D E rpoB gene 5 Probes bind to wild type Probes do not bind to mutant sequence 1 Probe for SPC (B. globigii) 6 fluorescent dyes detected simultaneously Molecular Beacon Target Hybrid We have developed a real-time PCR assay to detect rifampin resistance. Five molecular beacons have been designed to detect all mutations along the core region of the rpoB gene. PAGE | 16 16

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 | 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 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 These are all spiked buffer. Note also that the incidence of false positives is virtually nil over several years of development work in the Alland and Cepheid labs. PAGE | 19

Preliminary Performance Characteristics N.B. Data to be changed in final package insert PAGE | 20

Semi-quantitative results in Xpert MTB/RIF PAGE | 21

Amplification plot – TB positive/Rif sensitive MTB Positive Medium, Rif Resistance NOT DETECTED PAGE | 22

Amplification plots – TB positive/Rif resistance MTB Positive Low, Rif Resistance DETECTED PAGE | 23

Amplification plot – TB not detected MTB not detected PAGE | 24

TB confirmation and therapeutic guidance in less than 2 hours 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 | 25

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 PAGE | 26

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