Assessment of Clostridium difficile Infections by Quantitative Detection of tcdB Toxin by Use of a Real-Time Cell Analysis System
Assessment of Clostridium difficile Infections by Quantitative Detection of tcdB Toxin by Use of a Real-Time Cell Analysis System
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DOI：
10.1128/jcm.01104-10
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发表时间：
2010-11-01
影响因子：
9.4
通讯作者：
Tang, Yi-Wei
中科院分区：
文献类型：
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作者：
Ryder, Alex B.;Huang, Ying;Tang, Yi-Wei
我们探索了使用实时细胞分析（RTCA）系统，通过监测HS 27细胞对tcdB毒素的动态反应来评估人粪便标本中的艰难梭菌毒素。梭艰难梭菌毒素对细胞产生细胞毒性作用，导致细胞阻抗的剂量依赖性和时间依赖性降低。RTCA法对C.艰难梭菌毒素B与其他肠毒素无交叉反应;艰难梭菌或其他梭菌属物种。对300份连续采集的疑似C.艰难梭菌感染（CDI）。通过实时PCR检测（PCR）、谷氨酸脱氢酶和毒素A/B双酶免疫测定（EIA）和RTCA检测对每份粪便标本进行平行检测。与参比标准品相比，RTCA的特异性为99.6%，灵敏度为87.5%（28/32），高于EIA结果（P = 0.005），但低于PCR结果（P = 0.057）。此外，RTCA测定允许定量给定标本中的毒素蛋白浓度。在甲硝唑和/或万古霉素治疗前采集的RTCA阳性标本中，观察到毒素蛋白浓度与临床CDI严重程度之间存在显著相关性（R-2 = 0.732，P = 0.0004）。治疗后的毒素浓度（0.89 ng/ml）显著低于治疗前的毒素浓度（15.68 ng/ml，Wilcoxon P = 0.01）。本研究表明RTCA检测为C.艰难感染。
We explored the use of a real-time cell analysis (RTCA) system for the assessment of Clostridium difficile toxins in human stool specimens by monitoring the dynamic responses of the HS27 cells to tcdB toxins. The C. difficile toxin caused cytotoxic effects on the cells, which resulted in a dose-dependent and time-dependent decrease in cell impedance. The RTCA assay possessed an analytical sensitivity of 0.2 ng/ml for C. difficile toxin B with no cross-reactions with other enterotoxins, nontoxigenic C. difficile, or other Clostridum species. Clinical validation was performed on 300 consecutively collected stool specimens from patients with suspected C. difficile infection (CDI). Each stool specimen was tested in parallel by a real-time PCR assay (PCR), a dual glutamate dehydrogenase and toxin A/B enzyme immunoassay (EIA), and the RTCA assay. In comparison to a reference standard in a combination of the three assays, the RTCA had a specificity of 99.6% and a sensitivity of 87.5% (28 of 32), which was higher than the EIA result (P = 0.005) but lower than the PCR result (P = 0.057). In addition, the RTCA assay allowed for quantification of toxin protein concentration in a given specimen. Among RTCA-positive specimens collected prior to treatment with metronidazole and/or vancomycin, a significant correlation between toxin protein concentrations and clinical CDI severities was observed (R-2 = 0.732, P = 0.0004). Toxin concentrations after treatment (0.89 ng/ml) were significantly lower than those prior to the treatment (15.68 ng/ml, Wilcoxon P = 0.01). The study demonstrates that the RTCA assay provides a functional tool for the potential assessment of C. difficile infections.