MVZ Laboratory Dr. Limbach, Heidelberg, Germany.
Center for Experimental Medicine, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany.
J Microbiol Methods. 2020 May;172:105882. doi: 10.1016/j.mimet.2020.105882. Epub 2020 Feb 28.
The cobas® omni Utility Channel enables users to integrate lab-developed tests (LDTs) on the cobas® 6800 System to perform molecular diagnostics with high-throughput capacity and full automation. At present, there are no CE- or FDA-approved tests for stool pathogens on this system. To assess the performance of stool as a matrix, we evaluated the analytical and clinical performance of an LDT for detection of Clostridioides difficile (C. difficile) toxin B using the Utility Channel (C.diff_UTC).
A 10% stool suspension prepared from liquid stool samples diluted in phosphate buffered saline was used for analysis. Limit of detection (LoD) was determined in six dilutions with 126 replicates/dilution. Clinical evaluation was performed using 514 predetermined patient stool samples from two study sites in Germany. The C.diff_UTC was compared with LC 480 amplification and an LDT or the R-BioPharm C. difficile assay. Discrepant results were further analyzed using the GeneXpert C. difficile assay.
Limit of detection was 23.48 cfu/mL (95% Confidence Interval [CI]: 19.14-31.01) with inter-run variation of <2 cycle thresholds at 3 × and 10 × LoD. No cross-reactivity was observed with a panel of fecal organisms and pathogens. Bioinformatic analysis showed coverage of the major C. difficile toxinotypes by the primer/probe set. Clinical evaluation revealed sensitivity of 96.7% (95% CI: 88.7-99.6) and specificity of 99.3% (95% CI: 98.0-99.9) compared with the reference method; inhibition rate was 3.5% (18/514).
Using a predesigned primer/probe set, the C.diff_UTC assay features analytical performance and clinical sensitivity and specificity comparable to currently available nucleic acid amplification tests (NAATs) and is suitable for high-throughput testing. This was a proof-of-concept study, indicating the cobas Utility Channel could likely be adapted for other clinically relevant stool pathogens in outbreak scenarios.
cobas® omni 应用通道使用户能够在 cobas® 6800 系统上整合实验室开发的检测（LDT），从而以高通量和全自动化方式进行分子诊断。目前，该系统尚未获得用于粪便病原体的 CE 或 FDA 批准检测。为了评估粪便作为基质的性能，我们使用 Utility 通道（C.diff_UTC）评估了一种用于检测艰难梭菌（C. difficile）毒素 B 的 LDT 的分析和临床性能。
使用从磷酸盐缓冲盐水稀释的液体粪便样本制备的 10%粪便悬浮液进行分析。在六个稀释度下进行了 126 次重复/稀释度的检测限（LoD）确定。使用来自德国两个研究地点的 514 个预定患者粪便样本进行临床评估。C.diff_UTC 与 LC 480 扩增以及 LDT 或 R-BioPharm C. difficile 检测进行比较。使用 GeneXpert C. difficile 检测进一步分析了不一致的结果。
在 3×和 10×检测限下，检测限为 23.48 cfu/mL（95%置信区间 [CI]：19.14-31.01），运行间变异<2 个循环阈值。与粪便微生物和病原体的面板无交叉反应。生物信息学分析显示引物/探针组覆盖了主要的艰难梭菌毒素型。临床评估显示，与参考方法相比，该检测的敏感性为 96.7%（95% CI：88.7-99.6），特异性为 99.3%（95% CI：98.0-99.9）；抑制率为 3.5%（18/514）。
使用预设计的引物/探针组，C.diff_UTC 检测具有与现有核酸扩增检测（NAAT）相当的分析性能和临床敏感性及特异性，适用于高通量检测。这是一项概念验证研究，表明 cobas Utility 通道可能适合在爆发情况下用于其他临床相关的粪便病原体。
