High-Resolution Melting PCR as a Fast and Simple Molecular Biology-Based Method for the Identification of Hypervirulent Clostridioides difficile Strains Directly in Stool Samples

Abstract

Clostridioides difficile became one of the main causes of nosocomial infections in all clinical settings worldwide, especially among patients undergoing antibiotic therapy. The incidence and severity of C. difficile infections, from mild diarrhea to life-threatening pseudomembranous colitis, correlate with the spread of the hypervirulent binary toxin (CDT)-producing strains. The use of the real-time HRM-PCR method enables the identification of hypervirulent C. difficile strains directly in the diarrheal stool samples of patients suspected of being infected with this bacterium. For this purpose, the cdtA and cdtB genes encoding CDT subunits, as well as the species-specific gluD gene, were detected to identify the presence of this bacterium in the tested samples. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of the established method were also assessed. The obtained results were compared with the results of eazyplex® C. difficile complete test (AmplexDiagnostics GmbH) based on the LAMP method, used in standard microbiological diagnostics. The values of the assessed diagnostic parameters for the detected genes ranged from 58.82% to 98.85%. The lowest value (58.82%) was obtained for the PPV of cdtB and the highest (98.85%) for the NPV of this gene. The real-time HRM-PCR method enables fast and simple detection of the investigated genes of hypervirulent C. difficile strains and, after careful optimization, may demonstrate high potential for usefulness in routine microbiological diagnostics.