Increased environmental sample area and recovery of Clostridium difficile spores from hospital surfaces by quantitative PCR and enrichment culture.

OBJECTIVE: Clostridium difficile spores play an important role in transmission and can survive in the environment for several months. Optimal methods for measuring environmental C. difficile are unknown. We sought to determine whether increased sample surface area improved detection of C. difficile from environmental samples. SETTING: Samples were collected from 12 patient rooms in a tertiary-care hospital in Toronto, Canada. METHODS: Samples represented small surface-area and large surface-area floor and bedrail pairs from single-bed rooms of patients with low (without prior antibiotics), medium (with prior antibiotics), and high (C. difficile infected) shedding risk. Presence of C. difficile in samples was measured using quantitative polymerase chain reaction (qPCR) with targets on the 16S rRNA and toxin B genes and using enrichment culture. RESULTS: Of the 48 samples, 64·6% were positive by 16S qPCR (geometric mean, 13·8 spores); 39·6% were positive by toxin B qPCR (geometric mean, 1·9 spores); and 43·8% were positive by enrichment culture. By 16S qPCR, each 10-fold increase in sample surface area yielded 6·6 times (95% CI, 3·2-13) more spores. Floor surfaces yielded 27 times (95% CI, 4·9-181) more spores than bedrails, and rooms of C. difficile-positive patients yielded 11 times (95% CI, 0·55-164) more spores than those of patients without prior antibiotics. Toxin B qPCR and enrichment culture returned analogous findings. CONCLUSIONS: Clostridium difficile spores were identified in most floor and bedrail samples, and increased surface area improved detection. Future research aiming to understand the role of environmental C. difficile in transmission should prefer samples with large surface areas.

Comparison of qPCR versus culture for the detection and quantification of Clostridium difficile environmental contamination.

Contaminated surfaces serve as an important reservoir for Clostridium difficile transmission. Current strategies to detect environmental contamination of C. difficile rely heavily on culture, and often only indicate presence versus absence of spores. The goal of this study was to compare quantitative PCR (qPCR) to culture for the detection and quantification of C. difficile from inert surfaces. First, we compared the limit of detection (LOD) of a 16S rRNA gene and toxin B gene qPCR assay for detection of C. difficile in solution. Second, we compared the LODs of 16S rRNA gene qPCR versus culture for detection of C. difficile from surfaces. Solution experiments were performed by direct seeding of spores into neutralizing broth, whereas surface experiments involved seeding of spores onto plastic test surfaces, and recovery using sponge swabs. Both experiments were conducted using spores expressing short (NAP1) and long (NAP4) hair lengths. Combining data from both strains, the overall LOD for C. difficile cells in solution was 1.4 cells for 16S rRNA gene and 23.6 cells for toxin B gene qPCR (p<0.001). The overall LOD for C. difficile cells from surfaces was 17.1 cells for 16S rRNA gene qPCR and 54.5 cells for culture (p = 0.05), and was not statistically different between strains for each method (p = 0.52). Overall, the proportion of C. difficile cells recovered from surfaces was good when detected by 16S rRNA gene qPCR and culture (qPCR: 76%, culture: 67%, p = 0.36), but, 16S rRNA gene qPCR was capable of detecting lower levels of surface contamination. Future work attempting to measure the presence of C. difficile on environmental surfaces should consider using qPCR.