A commercial multiplex real-time PCR assay (Cepheid Xpert C. difficile assay) for the diagnosis of Clostridium difficile infection was evaluated. The sensitivity and specificity of the Cepheid assay were 97.1% and 93.0% for fresh stools, using the cell cytotoxicity neutralization assay as the reference. Using PCR ribotyping as the reference for ribotype 027 strains, the corresponding figures were 100% and 98.1%, respectively.Clostridium difficile infection (CDI) has increased in frequency and severity in North America and Europe over the last 5 years, largely due to the emergence of the epidemic PCR ribotype 027 strain (10, 11). The diagnosis of CDI is usually based on a clinical history of recent antimicrobial usage and diarrhea in combination with laboratory tests (9). Therefore, rapid and accurate microbiological diagnosis is urgently needed. The Cepheid Xpert C. difficile assay (Sunnyvale, CA) is a real-time multiplex PCR assay performed on the Cepheid GeneXpert Dx system. Proprietary primers specific for the toxin B gene (tcdB), binary toxin genes (cdtA and cdtB), and tcdC gene single-base deletion at nucleotide 117 were designed to detect toxigenic C. difficile and the presumptive PCR ribotype 027 strain. The purpose of this study was to evaluate the Cepheid Xpert C. difficile multiplex real-time PCR assay for the detection of toxigenic C. difficile strains and the presumptive ribotype 027.There were four serial investigations in the present study. In investigation 1, 205 frozen C. difficile strains collected during 2007 and 2008 were analyzed. In investigation 2, 195 frozen stool specimens belonging to different categories were selected based on direct cell cytotoxicity neutralization assay (CCNA) and toxigenic anaerobic culture results. Because PCR ribotype 027 is uncommon in Sweden, 40 frozen stool specimens collected in the United States were also analyzed. In investigation 3, 30 pairs of fresh-frozen stool specimens were analyzed. The fresh stool was analyzed within 24 h of collection, and then the leftover was stored at Ϫ20°C for 3 days and retested. In investigation 4, 220 consecutive fresh, unformed stool specimens (Bristol Stool Chart grade 5 to 7) from patients older than 2 years were analyzed within 24 h of collection. Eligible participants were those symptomatic patients who had a stool sample submitted to the Karolinska University Hospital for routine C. difficile testing.Unrepeated strains and stools were determined for C. difficile test by CCNA with a commercial C. difficile toxin/antitoxin kit (TechLab, Blacksburg, VA). For the stool specimens, anaerobic cultures on selective taurocholate cycloserine-cefoxitin-fructose agar plates were also performed (13). All isolates were typed by PCR ribotyping (19).Concurrently, the Cepheid Xpert C. difficile assay was performed according to protocols provided by the manufacturer. Each kit contained single-use disposable cartridges with integrated reaction chambers and reagents. A sterile Copan swab was dipped into the stool specimen or used to pick one...