Automated Detection of Toxigenic Clostridium difficile in Clinical Samples: Isothermal tcdB Amplification Coupled to Array-Based Detection

Clostridium difficile can carry a genetically variable pathogenicity locus (PaLoc), which encodes clostridial toxins A and B. In hospitals and in the community at large, this organism is increasingly identified as a pathogen. To develop a diagnostic test that combines the strengths of immunoassays (cost) and DNA amplification assays (sensitivity/specificity), we targeted a genetically stable PaLoc region, amplifying tcdB sequences and detecting them by hybridization capture. The assay employs a hot-start iso-thermal method coupled to a multiplexed chip-based readout, creating a manual assay that detects toxigenic C. difficilewith high sensitivity and specificity within 1 h. Assay automation on an electromechanical instrument produced an analytical sensi-tivity of 10 CFU (95 % probability of detection) of C. difficile in fecal samples, along with discrimination against other enteric bacteria. To verify automated assay function, 130 patient samples were tested: 31/32 positive samples (97 % sensitive; 95 % confi-dence interval [CI], 82 to 99%) and 98/98 negative samples (100 % specific; 95 % CI, 95 to 100%) were scored correctly. Large-scale clinical studies are now planned to determine clinical sensitivity and specificity. Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacterium. Infection by toxin-producing C. difficile causes a spectrum of disease from mild diarrhea to fulminant pseudomembranous colitis (4, 27). Although C. difficile is appar-ently an ancient species, having emerged more than 1 million years ago (18), it has been recognized as a human pathogen fo