评价 tcdB 实时 PCR 在产毒艰难梭菌三步诊断算法中的应用。
Evaluation of tcdB real-time PCR in a three-step diagnostic algorithm for detection of toxigenic Clostridium difficile.
机构信息
Clinical Microbiology Laboratory, Harborview Medical Center, Seattle, Washington 98104, USA.
出版信息
J Clin Microbiol. 2010 Jan;48(1):124-30. doi: 10.1128/JCM.00734-09. Epub 2009 Nov 18.
Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified "gold standard" that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of specimens, although this approach is less sensitive than performing DPCR on all specimens.
艰难梭菌是住院患者中最常见的传染性腹泻病因。目前对于产毒艰难梭菌的检测方法仍然存在争议，因为没有一种检测方法既敏感、特异又经济实惠。我们开发了一种实时 PCR 方法（直接粪便 PCR [DPCR]），可直接从粪便标本中检测编码毒素 B 的 tcdB 基因，并将其与酶联免疫吸附试验（EIA）结合，形成三步法方案。对 699 份经 Wampole C Diff Quik Chek EIA（GDH-Q）检测谷氨酸脱氢酶（GDH）阳性且经 Wampole Tox A/B Quik Chek EIA（AB-Q）检测毒素 A 和 B 均为阴性的标本进行了 DPCR 检测。该三步法方案的性能与改良的“金标准”（结合细胞毒性试验和 DPCR）进行了比较。我们还进行了一项单独的调查，以评估 GDH-Q 作为筛查试验的敏感性，结果在 211 份 GDH-Q 阴性标本中发现了 1.9%的产毒艰难梭菌。联合 GDH-Q、AB-Q 和 DPCR 的算法的总体敏感性、特异性和阳性与阴性预测值分别为 83.8%、99.7%、97.1%和 97.9%。而单独进行细胞毒性试验的结果分别为 58.8%、100%、100%和 94.9%。相比之下，使用相同的改良金标准，DPCR 的敏感性和特异性估计分别为 97.5%和 99.7%。细胞毒性试验和毒素 EIA 均不够敏感，无法排除产毒艰难梭菌，将 EIA 与细胞毒性试验联合应用于三步法方案也未能显著提高敏感性。DPCR 是一种用于检测产毒艰难梭菌的敏感、特异方法，其阳性检测结果的成本效益与其他方法相当，且能在同一天获得结果。在 GDH EIA 阳性、毒素 EIA 阴性标本中使用 DPCR 进行分析的三步法方案为 87.7%的标本提供了一种方便、特异且快速获得结果的替代方法，尽管该方法的敏感性不如对所有标本进行 DPCR 检测。
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