Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples

BACKGROUND: The cobas(R) omni Utility Channel enables users to integrate lab-developed tests (LDTs) on the cobas(R) 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). METHODS: 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. RESULTS: Limit of detection was 23.48cfu/mL (95% Confidence Interval [CI]: 19.14-31.01) with inter-run variation of <2cycle thresholds at 3xand 10xLoD. 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). CONCLUSION: 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.