Quantitative PCR Highly Sensitive for Detecting Surface C Difficile Contamination

In a study led by Laura K. MacDougall, of Public Health Ontario, in Toronto, Ontario, Canada, 16S rRNA gene quantitative polymerase chain reaction (qPCR) offered greater sensitivity than culture for detecting Clostridium difficile on environmental surfaces.

“The use of qPCR for detection of environmental C difficile has the potential to offer a number of advantages over culturing including reduced cost and turnaround time for results, as well as the ability to detect both culturable and nonculturable contamination,” the investigators wrote in their PLoS One study. “Therefore, in practice, both switching to 16s rRNA gene qPCR and increasing the sample surface area are strategies that are likely to yield higher probability of detecting spores.”

Investigators compared the limit of detection of a toxin B gene and 16S rRNA gene qPCR assay for C difficile detection. They seeded test surfaces with C difficile spores of short (NAP1) and long (NAP4) hair lengths. The limit of detections of 16S rRNA gene qPCR was compared with culture for C difficile from inert surfaces.

Cultures of C difficile were cultivated under anaerobic conditions, and Brucella supplemented agar (BSA) was used for 10 days to culture C difficile and induce sporulation. Solution experiments and surface experiments were performed by seeding spores into neutralizing broth and onto plastic test surfaces, respectively.

The combination of data from both strains yielded significantly lower C difficile limit of detection for 16S rRNA gene compared with toxin B gene qPCR (1.4 cells vs 23.6 cells, respectively; P <.001). Although the overall surfaces limit of detection for C difficile cells was greater for culture compared with 16S rRNA gene qPCR (54.5 cells vs 17.1 cells, respectively; P = .05), no significant difference was observed between strains based NAP1 vs NAP4 (P = .52).

A high proportion of C difficile cells were recovered from surfaces when detected using 16S rRNA gene qPCR and culture (76% and 67%, respectively), with no difference between either detection method (P = .36). The proportion of C difficile cells detected were also similar for NAP1 and NAP4 strains using qPCR (75% vs 77%; P = .87) and culture (76% vs 58%; P = .08).

Although 16S rRNA gene qPCR was able to detect low-level contamination, the identified strains may not be toxigenic, requiring further testing to determine clinical implications. “In the hospital setting, this may not pose a significant limitation since the majority of surface-isolated C difficile has been found to carry the tcdA and tcdB genes,” the investigators reported.

Findings of limit of detections from plastic surfaces from this study may not be generalizable to surfaces of other materials. In addition, further research that takes into account environmental cleaning effects or air exposure duration on culturability may be necessary.

“Future studies quantifying the environmental burden of C difficile should consider 16S rRNA gene qPCR for improved detection and enumeration,” the investigators concluded. “Our ability to recover spores of both types at similar efficiencies suggests similar adhesion between the 2 strains to plastic, but we cannot rule out the possibility of differences in adhesion to other surface materials or after longer periods post-deposition.”

The study, “Comparison of qPCR versus culture for the detection and quantification of Clostridium difficile environmental contamination,” was published in PLOS One.