Department of Pathology, Texas Children's Hospital, and Baylor College of Medicine, Houston, TX 77030, USA.
J Clin Microbiol. 2011 Mar;49(3):851-7. doi: 10.1128/JCM.01983-10. Epub 2011 Jan 5.
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.
艰难梭菌是医院获得性抗生素相关性感染性腹泻和伪膜性结肠炎的主要原因。艰难梭菌的检测已由厌氧细菌培养和/或细胞毒性测定法，很大程度上被快速酶免疫测定法（EIA）所取代。然而，由于粪便 EIA 的敏感性不足，我们开发了一种针对艰难梭菌毒素基因 tcdA 和 tcdB 的多重实时 PCR 检测方法。对疑似患有艰难梭菌相关性疾病的住院儿科患者的粪便标本进行前瞻性培养，在环丝氨酸-头孢西丁-果糖琼脂上进行，随后进行酒精冲击。评估了六种检测方式，包括粪便 EIA、培养 EIA 以及培养分离物和粪便标本的实时 PCR（tcdA 和 tcdB）。使用 tcdA 和 tcdB 基因特异性引物和水解探针，通过 LightCycler 平台（罗氏诊断公司，印第安纳波利斯，IN）进行实时 PCR 检测。对 96 例儿科患者的 157 个样本进行了分析。粪便实时 PCR 和粪便 EIA 的敏感性分别为 95%和 35%，两种方法的特异性均为 100%。粪便实时 PCR 的最低检测限为每个反应 30 CFU/ml 粪便样本的 tcdA 和 tcdB。本研究强调了粪便毒素 EIA 在儿科环境中的性能不佳。粪便样本中艰难梭菌毒素基因的实时 PCR 直接检测显示出优于粪便和培养 EIA 的敏感性，与培养分离物实时 PCR 检测的性能相当。粪便样本 DNA 的实时 PCR 是一种快速且具有成本效益的儿科诊断方式，应有助于进行适当的患者管理，并阻止 EIA 的连续检测实践。
