A new polymerase chain reaction (PCR) method is enabling investigators to detect clostridiodes difficile samples in fecal samples with high sensitivity, specificity, and repeatability.

A team, led by Xiao-xi Jia, State Key Laboratory Infections Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, tested the new detection method on a large sample of C difficile isolates and fecal samples.

The Technique

In the study, the investigators developed a new single-tube multiplex real-time PCR technique to enable the detection of toxigenic C difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets.

This could be performed on different kinds of PCR devices with improved detection efficiency, including point-of-care testing (POCT).

The team evaluated the specificity, sensitivity, and repeatability for each gene using 69 C difficile isolates and 74 fecal samples and then compared the results with established PCR, qPCR, and ELISA methods.

Interspecies specificity was entirely based on 6 common intestinal pathogens—Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum.

The lower detection limit for tcdA with pure C difficile DNA was 101 copies/μL, while the lower detection limit with pure C difficile DNA for tcdB, and cdtB was 100, and 100 copies/μL, respectively.

The coefficients of variation among the different experimental batches and within each experimental batch were both less than 3%.

This shows that the method has strong repeatability.

The lower detection limit of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively.

The efficiency for detection of tcdA compared to established PCR and real-time PCR method showed high consistency (98.4%) and similar sensitivity.

The team confirmed inconsistent results using ELISA and found identical results with the new method.

The sensitivity for detecting toxigenic C difficile in fecal samples were 96.49%, while the specificity was 94.12% compared with the toxigenic culture.

“This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China,” the authors wrote.

The study, “A rapid multiplex real-time PCR detection of toxigenic Clostridioides difficile directly from fecal samples,” was published online in 3 Biotech.