title: Evaluation of multiplex PCR with enhanced spore germination for detection of Clostridium difficile from stool samples of the hospitalized patients.
abstract: Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37 °C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.
authors.affiliation: Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Surang
authors.initials: S
authors.lastname: Chankhamhaengdecha
authors.forename: Piyapong
authors.initials: P
authors.lastname: Hadpanus
authors.forename: Amornrat
authors.initials: A
authors.lastname: Aroonnual
authors.forename: Puriya
authors.initials: P
authors.lastname: Ngamwongsatit
authors.forename: Darunee
authors.initials: D
authors.lastname: Chotiprasitsakul
authors.forename: Piriyaporn
authors.initials: P
authors.lastname: Chongtrakool
authors.forename: Tavan
authors.initials: T
authors.lastname: Janvilisri
mesh_terms: D016360:Clostridioides difficile / Q000302:isolation & purification* / Q000472:pathogenicity; D003428:Cross Infection / Q000175:diagnosis* / Q000382:microbiology; D003937:Diagnosis, Differential; D003967:Diarrhea / Q000175:diagnosis* / Q000382:microbiology; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis* / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D013171:Spores, Bacterial / Q000302:isolation & purification