title: Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile.
abstract: The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5 × 10(2) colony-forming units per 100 mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI.
authors.affiliation: National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan.
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Senoh
authors.forename: Haru
authors.initials: H
authors.lastname: Kato
authors.forename: Tomoko
authors.initials: T
authors.lastname: Murase
authors.forename: Hideharu
authors.initials: H
authors.lastname: Hagiya
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Tagashira
authors.forename: Tadashi
authors.initials: T
authors.lastname: Fukuda
authors.forename: Masaaki
authors.initials: M
authors.lastname: Iwaki
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Keigo
authors.initials: K
authors.lastname: Shibayama
mesh_terms: D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D016360:Clostridioides difficile / Q000235:genetics / Q000254:growth & development*; D005243:Feces / Q000382:microbiology; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D001427:Bacterial Toxins; D012333:RNA, Messenger
keywords: Clostridium difficile; Detection method; RT-PCR; Vegetative cells