Infection Control & Hospital Epidemiology 100,000 Positive 10,000 100 1,000 75 100 50 25 10 0


0 1


Negative


Enrichment culture result % Positive


921


0


10


100 1,000 10,000 100,000 Estimated spore count (16S)


0 1


10


100 Fig. 2. Association between the testing methods. Panel A: 16S qPCR and Toxin B qPCR. Panel B: 16S qPCR and enrichment culture.


C. difficile contamination, including culture,24 enrichment cul- ture,25 and qPCR.18 Culture-based techniques are commonly used and inexpensive, but they can be plagued by low detection limits, issues of nonculturability, and long turnaround times for results. Although qPCR is a more sensitive and culture-independent technique, it is also more labor intensive and costly, largely due to the DNA extraction step. However, it can produce results within 2–3 hours. qPCR is routinely used for clinical diagnostics and has also been adapted for environmental sampling.18 We found that 16S qPCR was more sensitive than toxin B qPCR. We also found that results from qPCR-based techniques and culture-based techniques were strongly correlated. Increasing levels of con- tamination by 16S qPCR were strongly associated with increased levels by toxin B qPCR and enrichment culture-based positivity. At levels beyond 1,000 spores by 16S qPCR, all toxin B qPCR samples and enrichment culture samples were positive, suggesting that toxin B qPCR and enrichment culture may have been negative due to a lack of sensitivity of the laboratory technique rather than a lack of toxigenic or live cells on those surfaces. Using real-world environmental samples, this study demon-


ques for measurement of environmental C. difficile. We also found that C. difficile contamination was common on bedrails and floors and was significantly more common in the rooms of patients with C. difficile infection. Several techniques have been used to measure environmental


Previous studies have shown that, for Bacillus atrophaeus, large surface-area sampling (1 m2) translates into greater sensitivity for detection and necessitates fewer samples for testing exposure– outcome associations, compared with samples with smaller surface areas.26 A more recent study used large surface area samples (up to 0.22m2) to quantify the microbial bioburden of several hospital pathogens.9 Our work showed that all techniques tested, including both culture and culture-independent techniques, reacted positively to increased sample size, though the clinical relevance of environmental C. difficile burden has not yet been established. We found that C. difficile contamination was ubiquitous in the


strated that large surface-area samples yielded higher positivity and higher counts of spores across several different micro- biologic techniques and 2 surface types. These findings provide further evidence of the advantages of maximizing surface area when conducting environmental sampling. For 16S qPCR, the effect of surface area on C. difficile capture may have been moderately stronger among floor samples than among bedrail samples. This finding may have been due to inconsistencies in the sampling of bedrails because bedrails have complex surfaces.


hospital rooms we sampled. All rooms, even those of patients without a history of C. difficile or antibiotic use, yielded positive samples by 16S qPCR. It has been shown that floor-surface samples are approximately twice as likely to be culture positive as bedrail samples,6 and surfaces more proximate to patients are more likely to be contaminated than those farther from patients.27 In our study, floor surfaces actually had 10 times more C. difficile contamination than bedrail surfaces. Also, 100% of the 12 large floor samples we collected were contaminated with C. difficile according to 16S qPCR. Clostridium difficile on floor surfaces may be a risk to patients because microbes on the floor may be transported within wards by shoe soles28 are continually resus- pended by foot traffic.29 Furthermore, mobile patients can con- taminate linens and themselves via their feet or shoes.13 Although our results do not allow us to assess directionality, the finding of strong relatedness of strains recovered within wards suggests that spatial dissemination involving floors may be a concern. These results also lend support to several studies that have shown the importance of ward-level effects for the transmission of C. difficile and other hospital-associated infections.30–32 The infective dose of C. difficile for a healthy but susceptible


inpatient is not known33 and as such, safe levels of environmental C. difficile are not known. For mice, the environmental infectious


1,000 10,000 100,000 Estimated spore count (16S)


Estimated spore count (Toxin B)


Enrichment culture result (% positive)


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