National Reference Laboratory for C. difficile, Paris VI University, Paris, France.
Eur J Clin Microbiol Infect Dis. 2011 Oct;30(10):1279-85. doi: 10.1007/s10096-011-1224-z. Epub 2011 Apr 13.
The gold standards for the diagnosis of Clostridium difficile infections (CDIs) are the cytotoxicity assay and the toxigenic culture. However, both methods are time-consuming and the results are not available before 24-48 h. We developed and evaluated a multiplex in-house real-time polymerase chain reaction (PCR) assay for the simultaneous detection of toxigenic strains of C. difficile and the presumptive identification of the epidemic NAP1/027/BI strain from stools. Amplifications were performed using specific primers for tcdB and tcdC on an ABI Prism 7300 (Applied Biosystems). The detection of amplicons was done using TaqMan probes. The analytical sensitivity of the multiplex real-time PCR for detecting tcdB was estimated to 10 CFU/g of stools. This assay was assessed from 881 consecutive unformed stools from patients suspected of having CDI. The gold standard was the toxigenic culture for the diagnosis of CDI and PCR ribotyping for the identification of the NAP1/027/BI strain. The prevalence of positive toxigenic culture was 9.31%. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 86.59%, 97.43%, 78.02%, and 98.57%, respectively, for the real-time PCR and 70.73%, 100%, 100%, and 97.08%, respectively, for the cytotoxicity assay.
艰难梭菌感染 (CDI) 的诊断金标准是细胞毒性测定和产毒培养。然而，这两种方法都很耗时，结果要在 24-48 小时后才能得到。我们开发并评估了一种用于从粪便中同时检测产毒艰难梭菌菌株和假定的 NAP1/027/BI 流行株的内部多重实时聚合酶链反应 (PCR) 检测方法。使用针对 tcdB 和 tcdC 的特异性引物在 ABI Prism 7300（Applied Biosystems）上进行扩增。使用 TaqMan 探针检测扩增子。用于检测 tcdB 的多重实时 PCR 的分析灵敏度估计为 10 CFU/g 粪便。该检测方法评估了 881 例来自疑似 CDI 患者的未成形粪便。金标准是用于诊断 CDI 的产毒培养和用于鉴定 NAP1/027/BI 菌株的 PCR 核糖体分型。阳性产毒培养的患病率为 9.31%。与产毒培养相比，实时 PCR 的敏感性、特异性、阳性预测值和阴性预测值分别为 86.59%、97.43%、78.02%和 98.57%，细胞毒性测定分别为 70.73%、100%、100%和 97.08%。
