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Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection ofC. difficile by 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. difficile toxin genestcdA andtcdB . Stool samples from hospitalized pediatric patients suspected of havingC. difficile -associated disease were prospectively cultured on cycloserine-cefoxitin-fructose agar following alcohol shock. Six testing modalities were evaluated, including stool EIA, culture EIA, and real-time PCR (tcdA andtcdB ) of cultured isolates and stool samples. Real-time PCR detection was performed withtcdA andtcdB gene-specific primers and hydrolysis probes using the LightCycler platforms (Roche Diagnostics, Indianapolis, IN). A total of 157 samples from 96 pediatric patients were analyzed. The sensitivities of stool real-time PCR and stool EIA were 95% and 35%, respectively, with a specificity of 100% for both methods. The lower limit of detection of the stool real-time PCR was 30 CFU/ml of stool sample per reaction fortcdA andtcdB . This study highlights the poor performance of stool toxin EIAs in pediatric settings. Direct detection ofC. difficile toxin genes in stool samples by real-time PCR showed sensitivity superior to that of stool and culture EIAs and performance comparable to that of real-time PCR assay of cultured isolates. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for children that should facilitate appropriate patient management and halt the practice of serial testing by EIA.

Rapid Stool-Based Diagnosis of Clostridium difficile Infection by Real-Time PCR in a Children's Hospital