Presented by Dr. Brecher at the 40th Annual Symposium "Diagnostic and Clinical Challenges of 20th Century Microbes", held on Nov 18, 2010 in Philadelphia.

1. Overview of Clostridium Difficile Infections (CDI) Stephen M. Brecher Ph.D. VA Boston HealthCare System BU School of Medicine

2. The opinions expressed in this presentation are those of the presenter and do not necessarily represent the views of the Veterans Affairs Health- Care System

13. Pathogenesis of CDI Asymptomatic C. difficile colonization C. difficile exposure Antimicrobial C. difficile infection Hospitalization From Johnson S, Gerding DN. Clin Infect Dis. 1998;26:1027-1036; with permission.

16. In Vitro Production of Toxins in Epidemic Strain From Warny M, et al. Lancet . 2005;366:1079-1084; with permission.

21. Laboratory Diagnosis of CDI Laboratory Diagnosis Enzyme Immunoassay (EIA) Glutamate Dehydrogenase (GDH) Cell Culture Neutralization Assay (CCNA) Toxigenic Culture (Culture and CCNA) Molecular Based (PCR Or LAMP) Stool Culture

23. “Plate Sin With Gold and it Goes Untarnished”

24. Cell Culture Neutralization Assay Performance Revisited * Compared to toxigenic culture. + Compared to clinical case definition and multiple test methods. Slide provided by Dr. Karen Carroll Study Method Sensitivity (%) Specificity (%) Eastwood, et al. JCM 2009; 47:3211 In house Vero cell assay* 86.4 99.2 Barbut, et al. JCM 2009;47:1276 In house MRC-5 cells* 75.8 100 Stamper, et al JCM 2009;47:373 TechLab TOX-B test* 67.2 99.1 Peterson, et al. CID 2007;45:1152 In house MRC-5 cells + 76.7 97.1

29. BD GeneOhm™ Cdiff Assay Procedure Overview Definitive On-screen Results Results in <2 Hours Stool Specimen Slide courtesy BD GeneOhm lideS provide Specimen Preparation Lysis - DNA Extraction Reconstitution Of Reagents Real-time PCR Analysis on the SmartCycler ®

30. pro GASTRO ™ Cd Workflow Specimens Clarified Stool 110 µl of nucleic acid for analysis Add buffer, Vortex & spin Slide courtesy Gen-Probe Dilute Internal Control. Combine 20 µl clarified stool and 10 µl Internal Control Run Lysis, Add Beads Perform extraction and elute in 110 µl

31. pro GASTRO ™ Cd Workflow Hands-on Time = 57 min Hands-off Time = 137 min TOTAL TIME = 194 min Slide courtesy Gen-Probe Extracted nucleic acid Prepare controls Combine 5 µl of nucleic acid & 20 µl of Mastermix in reaction tube Insert each reaction tube in SmartCycler Program instrument and run assay Mastermix

32. Xpert ® C. difficile PCR Test for Clostridium difficile Toxin B Only Results in an early as 30 minutes; assay complete in 45 minutes If negative

35. Summary of C. difficile PCR Published Data Slide courtesy of Dr. Susan Novak-Weekley Publication PCR Assay Sens/Spec Terhes, 2009 BD vs. Tox Culture* 96%/99% Not all negatives specimens tested by culture*. Stamper, 2009 BD vs. Tox Culture 83.6%/98.2% Eastwood, 2009 BD vs. CCCN BD vs. Tox Culture 92.2%/94% 88.5%/95.4% Kvach, 2010 BD vs. Tox Culture* 91.4%/100% Not all negatives specimens tested by culture*. Stamper, 2009 Prodesse vs. Tox Culture 77.3%/99.2% Huang, 2009 Cepheid Xpert vs. CCCN 97.1%/93.9% Novak-Weekley, 2010 Cepheid Xpert vs. Tox Culture 94.4%/96.3% Swindells, 2010 Cepheid Xpert vs Tox Culture BD vs Tox culture 100%/99.2% 94.4%/99.2%, Small “N”