Evaluation of Multiplex PCR with Enhanced Spore Germination for Detection ofClostridium difficilefrom Stool Samples of the Hospitalized Patients

Abstract

Clostridium difficileposes as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detectC. difficiledirectly 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 variousC. difficileribotypes, otherClostridiumspp., and non-Clostridiumstrains 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 ofC. difficilein 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.