title: A multiplex, internally controlled real-time PCR assay for detection of toxigenic Clostridium difficile and identification of hypervirulent strain 027/ST-1.
abstract: The purpose of this study was to validate a multiplex real-time PCR assay capable of detecting toxigenic Clostridium difficile and simultaneously identifying C. difficile ribotype 027/ST-1 by targeting the toxin genes tcdA, tcdB and cdtA in one reaction and in a separate reaction identifying the Δ117 deletion in tcdC associated with ribotype 027/ST-1. PCR was done prospectively on 704 samples routinely submitted to our department and results were compared to results of toxigenic culture. Sequencing of tcdC, multi locus sequence typing (MLST) and PCR ribotyping were done on cultured isolates to confirm the correct identification of the Δ117 deletion in tcdC and C. difficile ribotype 027/ST-1, respectively. The PCR assay displayed a sensitivity, specificity, PPV and NPV of 99.0%, 97.4%, 87.4% and 99.8%, respectively, compared to toxigenic culture on 665 samples evaluable both by PCR and culture. Sequencing of tcdC, ribotyping and MLST of cultured isolates validated the genotyping assay and confirmed the ability of the assay to correctly identify C. difficile ribotype 027/ST-1 in our current epidemiological setting. We describe the use of a combination of two separate PCR assays for sensitive and specific detection of toxigenic C. difficile and presumptive identification of C. difficile 027/ST-1.
authors.affiliation: Department of Clinical Microbiology 445, Hvidovre Hospital, Kettegaard Allé 30, DK-2650, Hvidovre, Denmark.
authors.forename: A M
authors.initials: AM
authors.lastname: Hoegh
authors.forename: J B
authors.initials: JB
authors.lastname: Nielsen
authors.forename: A
authors.initials: A
authors.lastname: Lester
authors.forename: A
authors.initials: A
authors.lastname: Friis-Møller
authors.forename: K
authors.initials: K
authors.lastname: Schønning
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D003015:Clostridium Infections / Q000175:diagnosis* / Q000382:microbiology*; D004768:Enterotoxins / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D058885:Multilocus Sequence Typing; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D021521:Ribotyping; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium