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Updated: May 5, 2026
A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment
Published on: May 25, 2017
High-Resolution Melting PCR as a Fast and Simple Molecular Biology-Based Method for the Identification of
Tomasz Bogiel1,2,3, Robert Górniak2,4, Weronika Ambroziak3
1Microbiology Department Ludwik Rydygier, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, 9 Maria Skłodowska-Curie Street, 85-094 Bydgoszcz, Poland.
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.
Insights
A new real-time HRM-PCR method quickly identifies hypervirulent Clostridioides difficile strains in patient stool samples. This technique aids in diagnosing C. difficile infections, particularly those caused by toxin-producing strains.
Area of Science:
- Medical Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Clostridioides difficile is a leading cause of hospital-acquired infections globally.
- Hypervirulent strains producing binary toxin (CDT) increase infection severity.
- Rapid diagnostics are crucial for managing C. difficile infections.
Purpose of the Study:
- To develop and evaluate a real-time HRM-PCR assay for detecting hypervirulent C. difficile.
- To assess the diagnostic accuracy of the assay in identifying CDT-producing strains.
- To compare the assay's performance against a standard LAMP-based diagnostic test.
Main Methods:
- Real-time high-resolution melting-PCR (HRM-PCR) targeting cdtA, cdtB, and gluD genes.
- Direct detection in patient diarrheal stool samples.
- Comparison with eazyplex® C. difficile complete test (LAMP method).
Main Results:
- The HRM-PCR method successfully detected target genes in C. difficile strains.
- Diagnostic parameters (sensitivity, specificity, PPV, NPV) varied by gene, with NPV for cdtB reaching 98.85%.
- The method demonstrated fast and simple detection of hypervirulent C. difficile genes.
Conclusions:
- Real-time HRM-PCR offers a rapid and straightforward approach for identifying hypervirulent C. difficile.
- The assay shows potential for integration into routine microbiological diagnostics.
- Further optimization may enhance its utility in clinical settings.
