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, PAGE | 1 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 PAGE | 2 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 PAGE | 3 Mixture is delivered to integrated reaction tube for nested real-time amplification and detection Total time to result = 1h45min GX-XVI system PAGE | 4 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 PAGE | 5 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: PAGE | 6 Alpha Trial TB Detection for Individual Patients Per patient analysis for Mtb detection TB CASE DETECTION TOTAL 95% CI PAGE | 7 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 PAGE | 8 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 PAGE | 9 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 PAGE | 10