A New Lab Developed Real Time PCR Assay for Direct Detection of C. Difficle from Stool Sample without DNA Extraction.
Clostridium difficileis a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection ofC. difficileby anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting theC. difficiletoxin genestcdB. stool samples from hospitalized pediatric patients suspected of havingC. difficile-associated disease were prospectively collected. Three testing modalities were evaluated, including enriched culture, cepheid Xpert and real-time Pcr (tcdB) on stool samples performed withtcdBgene-specific primers and hydrolysis probes. A total of 150 de-identified clinical specimen were analyzed. The sensitivities of stool real-time Pcr were 95% against cepheid Xpert C. difficile and 93% against enriched culture respectively, with a specificity of 97% and 94%. The lower limit of detection of the stool real-time PCR was 0.5 cFU/ml of per reaction fortcdB. Direct detection ofC. difficiletoxin genes in stool samples by real-time Pcr showed performance comparable to enriched culture. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for patients that should facilitate appropriate patient management.
Sino US Gene, Inc. 1452 #C, West Holt Ave. Pomona, CA 91768, USA.
Brandon
B
Li
Clostridium difficile; enzyme immunoassays; real-time PCR; tcdB