Multiple cross displacement amplification (MCDA) for rapid detection of toxigenic Clostridioides difficile as a...

Toxigenic Clostridioides difficile is the cause of C. difficile infection, highlighting the critical need for rapid and easy-to-use detection. In this study, a multiple cross displacement amplification (MCDA) assay was developed to detect toxigenic C. difficile and evaluated against real-time PCR and VIDAS C. difficile toxin A & B assay (CDAB). The results showed that MCDA targeting the C. difficile tcdB gene had a limit of detection (LOD) of 12.5 fg (2.5 copies of genomic DNA) per reaction with no non-specific amplification from other pathogens. The LOD for MCDA was the same as real-time PCR but had significantly faster detection time, detecting 500 pg and 12.5 fg per reaction in 5.7 ± 0.5 and 21.35 ± 2.2 min, respectively. Among 201 stool samples, MCDA identified 40 positive for toxigenic C. difficile with a shorter average detection time of 16.08 ± 4.62 min compared to real-time PCR (39.82 ± 4.44 min). Using cell cytotoxicity neutralization assays as reference, MCDA demonstrated a significantly higher specificity (92.4%) and a positive predictive value (67.5%) than real-time PCR (80.8 and 43.2%, respectively; P < 0.05). MCDA also had a significantly higher sensitivity (93.1%) and a negative predictive value (98.8%) compared to VIDAS CDAB (34.5 and 90.1%, respectively; P ≤ 0.001). This was also higher compared to real-time PCR (86.2 and 97.2%, P > 0.05). MCDA offers a promising screening alternative for point-of-care testing to detect toxigenic C. difficile and effectively rule out non-toxigenic C. difficile. It is rapid, easy to use, and has a balanced performance, making it applicable in clinical and community settings, particularly in resource-limited settings.IMPORTANCERapid detection of toxigenic Clostridioides difficile is important for early intervention and outbreak control of C. difficile infection in primary healthcare facilities. This study developed an MCDA assay and showed that it was rapid, sensitive, user-friendly, and a suitable point-of-care screening alternative for accurately detecting toxigenic C. difficile. MCDA had the same sensitivity as real-time PCR while providing significantly faster turnaround times and improved specificity along with positive and negative predictive values. Its balanced performance and rapid detection capability make it well-suited for clinical detection of toxigenic C. difficile and point-of-care testing in communities, particularly in resource-limited settings, and for potential self-testing.