Rapid Simultaneous Molecular Stool-Based Detection of Toxigenic Clostridioides difficile by Quantitative TaqMan Real-Time PCR Assay

Background: Clostridioides difficile is a major cause of nosocomial infectious diarrhea in hospitalized patients throughout the world. Methods: A multiplex real-time PCR assay was developed and evaluated in comparison with toxigenic culture (TC) (as gold standard method) for direct detection of toxigenic C. difficile in fecal specimens. The multiplex real-time PCR assay simultaneously detected glutamate dehydrogenase (gluD), toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtB) genes in stool samples. Results: The results of multiplex real-time PCR were compared to those of the TC method in 250 patients suspected of C. difficile infection. The prevalence of positive TC was 13.6%. Forty-two stool samples (16.8%) were determined to be gluD(+) using multiplex real-time PCR These included 35 (83.3%) toxigenic (32 tcdA(+), tcdB and three tcdW) and 7 (20.0%) were cdtB(+). The multiplex real-time PCR assay had a sensitivity of 91.45%, specificity of 99.54%, and positive and negative predictive values of 97% and 98.6%, respectively, compared to the TC method for diagnosis of C. difficile. The analytical sensitivity of the multiplex real-time PCR assay was estimated to be 10(2) CFU/g of stools and 0.0200 pg of genomic DNA from culture. The analytical specificity was determined to be 100% by using enteric and non-C. difficile standard bacterial strains. Conclusions: The molecular method developed in the study was rapid, sensitive, and specific for detection of toxigenic C. difficile. It is applicable to be performed in clinical laboratories and correlated well with the results obtained by TC.