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

Abstract

Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. 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 the C. difficile toxin genes tcdA and tcdB. Stool samples from hospitalized pediatric patients suspected of having C. 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 and tcdB) of cultured isolates and stool samples. Real-time PCR detection was performed with tcdA and tcdB 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 for tcdA and tcdB. This study highlights the poor performance of stool toxin EIAs in pediatric settings. Direct detection of C. 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.

Evolution of Testing Algorithms at a University Hospital for Detection of Clostridium difficile Infections

“…First, the increase in the number of CDI cases after the institution of the GDH-toxin A/B ICA-PCR algorithm was based on infection prevention chart review and not strictly laboratory findings, although the latter are used as part of the CDI case definition. Our findings are consistent with those of others who observed an increase in the number of specimens positive for C. difficile after the initiation of molecular testing (7,14). Second, we have strictly enforced rules in place in our laboratory that allow only the testing of diarrheic stool samples and no testing of cure stool specimens without consultation with a laboratory director (4).…”

“…The presence of toxigenic C. difficile can be determined by toxin enzyme immunoassay (insensitive), cytotoxin neutralization (CTN; relatively sensitive), DNA amplification (most sensitive but perhaps less specific), or toxigenic culture (most sensitive but quite slow, which makes it impractical for diagnostic purposes) (1,2,17,26). The second approach is to directly test stool samples for the presence of toxigenic C. difficile by using any of four commercially available, FDA-approved nucleic acid amplification techniques (NAATs) (3,5,9,10,11,12,13,14,22,23,26).…”

Clostridium difficile Infection in Infants and Children

“…24 In a recent study, the sensitivities of the real-time polymerase chain reaction (PCR) assay for toxin A/B compared with EIA for toxin A/B were superior (95% vs 35%, respectively), and the specificity was equal (100%). 25 With the use of the PCR, the positivity rates for stool samples doubled, from 7.9% to 8.3% with EIA to 14.9% to 18.1% with PCR, and the numbers of repeated samples decreased. Many children' s hospitals are converting to NAAT technology to diagnose CDIs, but more data are needed before NAATs can be used routinely.…”

ProbioticSaccharomyces boulardiiCNCM I-745 prevents outbreak-associatedClostridium difficile-associated cecal inflammation in hamsters

“…The DNA concentration was measured by a Nanodrop machine and was then normalized to 100 ng per PCR reaction with water. The PCR detection of tcdA and tcdB was performed in separate reactions as previously described (34). All sample reactions were performed in duplicate.…”