Objectives

To improve the diagnostic accuracy of Clostridioides difficile infection, current U.S. and E.U. guidelines recommend multistep testing that detects the presence of C. difficile and toxin in clinically relevant stool samples to confirm active disease. An accepted gold standard to detect C. difficile toxins is the cell cytotoxicity neutralization assay (CCNA). Although highly sensitive, the traditional CCNA has limitations. One such limitation is the subjective interpretation of an analyst to recognize cytopathic effects in cultured cells exposed to a fecal sample containing toxin. To overcome this limitation, an automated CCNA was developed that replaces most human pipetting steps with robotics and incorporates CellTiterGlo® for a semi-quantitative, non-subjective measure of cell viability instead of microscopy.

Methods

To determine sample positivity and control for non-specific cytopathic effects, two thresholds were defined and validated by evaluating the sample with/without antitoxin antisera (sample-antitoxin/sample + antitoxin): 1) a >70% cell viability threshold was validated with samples containing anti-toxin, and 2) a >1.2-fold difference cut-off where sample results above the cut-off are considered positive.

Results

Assay validation demonstrated excellent accuracy, precision, and sample linearity with an LOD of 126.9 pg/mL toxin-B in stool. The positivity cut-offs were clinically validated by comparing 322 diarrheal stool sample results with those run in a predicate, microscopic readout-based CCNA. The automated CCNA demonstrated 96% sensitivity and 100% specificity compared with the predicate CCNA. Conclusions: Overall, the automated CCNA provides a specific, sensitive, and reproducible tool to support determination of CDI epidemiology or the efficacy of interventions such as vaccines.

中文翻译：

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用于检测临床相关粪便样本中艰难梭菌毒素的自动化细胞细胞毒性中和试验的开发和临床验证

目标

为了提高艰难梭菌感染的诊断准确性，当前的美国和欧盟指南建议进行多步检测，检测临床相关粪便样本中艰难梭菌和毒素的存在，以确认活动性疾病。公认的检测艰难梭菌的金标准毒素是细胞毒性中和试验（CCNA）。虽然高度敏感，但传统的 CCNA 有局限性。一个这样的限制是分析人员的主观解释，以识别暴露于含有毒素的粪便样品的培养细胞中的细胞病变效应。为了克服这一限制，开发了一种自动化的 CCNA，它用机器人技术取代了大多数人工移液步骤，并结合了 CellTiterGlo®，用于细胞活力的半定量、非主观测量，而不是显微镜检查。

方法

为了确定样品阳性和非特异性细胞病变效应的对照，通过评估含有/不含抗毒素抗血清的样品（样品-抗毒素/样品 + 抗毒素）定义和验证了两个阈值：1) >70% 的细胞活力阈值用含有抗毒素的样品，以及 2) >1.2 倍的差异临界值，其中高于临界值的样本结果被认为是阳性的。

结果

测定验证证明了出色的准确度、精密度和样品线性，粪便中毒素 B 的 LOD 为 126.9 pg/mL。通过将 322 份腹泻粪便样本结果与在谓词、基于显微读数的 CCNA 中运行的结果进行比较，临床验证了阳性截止值。与谓词 CCNA 相比，自动化 CCNA 表现出 96% 的敏感性和 100% 的特异性。结论：总体而言，自动化CCNA提供了一种特异性、敏感和可重复的工具，以支持确定CDI流行病学或疫苗等干预措施的有效性。