Evaluation of Various Laboratory Methods for the Diagnosis of Clostridium difficile Infection Yousun Chung1, Ji Yeon Sung1, Ho Eun Chang2, Soon Hee Choi2, Yun Ji Hong1, Taek Soo Kim1,2, Kyoung Un Park1,2, Junghan Song1,2, Eui Chong Kim1 1Department of Laboratory Medicine, Seoul National University College of Medicine; 2Department of Laboratory Medicine, Seoul National University Bundang Hospital

Clostridium difficile Infection (CDI) BACKGROUND Clostridium difficile Infection (CDI) Leading cause of health care-associated diarrhea Clinical features overlapping with other antibiotic-associated diarrhea Increase in the number of cases and in the severity Emergence of hypervirulent toxigenic strain, BI/NAP1/027 Prompt CDI identification to prevent disease progression and to reduce additional nosocomial cases Lack of rapid, accurate, and inexpensive diagnostic test

Laboratory Diagnosis of CDI BACKGROUND Laboratory Diagnosis of CDI Culture Labor-intensive, long TAT Enzyme immunoassay (EIA) Easy, rapid TAT, variable performance Cell cytotoxicity neutralization assay (CCNA) Labor-intensive, long TAT, Expensive cell lines, Interpretation Molecular-based assays High sensitivity and specificity, short TAT, Expensive

Aims Compare the performance of different laboratory methods for the diagnosis of CDI Evaluate real-time PCR assays in diagnosis of CDI

Materials and Methods Materials Laboratory diagnostic methods 188 stool specimens from Seoul National University Bundang Hospital 52 C. difficile colonies: randomly selected Laboratory diagnostic methods chromID C. difficile (bioMérieux) Cell cytotoxicity neutralization assay (CCNA) VIDAS C. difficile Toxin A & B (bioMérieux) RIDA QUICK C. difficile Toxin A/B (R-Biopharm) RIDA SCREEN C. difficile Toxin A/B (R-Biopharm) RIDA GENE C. difficile & Toxin A/B (R-Biopharm) IMDx C. difficile (Abbott)

chromID C. difficile (bioMérieux) MATERIALS and METHODS chromID C. difficile (bioMérieux) Detection of β-glucosidase producing C. difficile strains Typical grey to black color of colonies VIDAS C. difficile Toxin A & B (bioMérieux) Enzyme linked fluorescent immunoassay Qualitative detection of C. difficile toxin A/B

Cell cytotoxicity neutralization assay (CCNA) MATERIALS and METHODS Cell cytotoxicity neutralization assay (CCNA) Inoculation of stool sample onto cultured human fibroblasts Reading in 2 hrs, 6 hrs, 24hrs, and 48hrs Cytopathic effect of toxin causes the fibroblasts to round up Absence of cytopathic effect with the anti-toxin Considered as a gold standard for C. difficile toxin Labor-intensive, long TAT, Expensive cell lines, Interpretation

RIDASCREEN® C. difficile Toxin A/B (R-Biopharm) MATERIALS and METHODS RIDA®QUICK C. difficile Toxin A/B (R-Biopharm) Immunochromatographic rapid assay Determination of Toxin A and Toxin B with anti-Toxin A and anti-Toxin B antibodies RIDASCREEN® C. difficile Toxin A/B (R-Biopharm) Enzyme immunoassay Detection of both toxin A/B with monoclonal antibodies

RIDA®GENE C. difficile & Toxin A/B (R-Biopharm) MATERIALS and METHODS RIDA®GENE C. difficile & Toxin A/B (R-Biopharm) Real-time PCR Detection of C. difficile (16S rRNA) and toxin (tcdA/tcdB genes) IMDx C. difficile (Abbott) Real-time PCR Detection of conserved regions of tcdA and tcdB genes

CCNA as the gold standard RESULTS CCNA as the gold standard chromID VIDAS RIDA QUICK SCREEN GENE IMDx Sensitivity Stool 79.1 74.6 10.4 47.8 89.6 96.9 Colony 75 12.5 59.4 Specificity 23.8 69 100 95.2 76.2 76.9 57.9 94.7 89.5 68.4 63.2 PPV 62.4 79.4 94.1 85.7 87.5 80 90.5 83.8 81.6 NPV 41.7 63 41.2 53.3 82.1 93.8 39.1 56.7 92.9 92.3

Real-time PCR assays as the gold standard RESULTS Real-time PCR assays as the gold standard chromID VIDAS RIDA QUICK SCREEN CCNA Sensitivity Stool 74.3 65.7 10.0 45.7 89.1 Culture 68.4 13.2 55.3 Specificity 15.9 56.8 100.0 95.5 84.2 50.0 92.9 PPV 58.4 70.7 94.1 90.5 78.8 NPV 28.0 51.0 41.1 52.5 82.1 36.8 29.8 43.3

Conclusions Enzyme immunoassay methods showed variable performance VIDAS showed relatively high sensitivity Real-time PCR methods showed high sensitivity and specificity Do not detect the presence of toxin itself Difficult choices to balance test performance and cost chromID + VIDAS → Molecular assay