1410
washing were present before washing and were not a result of contamination during the wash cycle.
Discussion
The aim of comparing recovery methods of C. difficile spores in the presence of soiling was to ascertain an efficient method of recovering spores in heavily soiled conditions often found on bed sheets of CDI patients. Because the vortexing method recovered significantly more spores than the stomaching method, this method was subsequently used in the laundering investigations. Several agitation methods were previously tested (data not shown) and were not as effective as vortexing. The vortexing method has also been used in other studies to assess the efficacy of the minimum laundry conditions required by HTM 01-04.13–15 Notably, the current healthcare laundry policy, HTM 01-04,7 suggests the use of ‘agitation’ to recover B. cereus spores for annual seasonal microbiological assessments. Stomaching for 1 minute was recommended by the laboratory responsible for microbiology at the commercial laundry in this study (personal communication, 2011). Therefore, the reported proportions of viable spores (B. cereus or C. difficile) recovered are dependent upon the type and duration of agitation method chosen by the testing laboratory. The use of varying recovery methods for spores from textiles between organizations is concerning because spore recovery can vary significantly depending on the spore recovery method. The test wash cycles used in this investigation all reached 75°C
and were held at ≥71°C for >3 minutes (+8 minutes mixing time), as required by thermal disinfection conditions of the policy HTM 01-04. For both test strains, the use of detergent was an important factor in significantly reducing the numbers of C. difficile spores (Fig. 2). We observed a >6 log10 reduction in the number of viable spores recovered from swatches after washing using the industrial detergent system in the simulated HTM 01- 04–compliant healthcare cycle. The >6 log10 reduction still applied after considering that the spore recovery method only recovered an average of 31% of the viable spores remaining on the swatch. In the control cycle without the industrial detergent system, thermal disinfection and agitation were found to be inadequate to affect a >5 log10 reduction in C. difficile spores.
Cross contamination occurred in both the control and deter-
gent washes, where spores removed from the inoculated swatches were transferred to other material present in the drum (Fig. 2). Cross contamination was significantly higher when the detergent system was not included in the cycle. Chemo-thermal disinfection proved to be significantly better
at decontaminating the swatches than thermal disinfection alone, but even in an optimized wash, 0.1–1 log10 cfu/25cm2 remained. This quantity would fail the microbiological validation require- ment of <100 cfu per test swatch with no pathogenic bacteria present. The SEM images in this study (Fig. 3) confirmed the presence of C. difficile spores on swatches after a 71°C wash with detergent. The healthcare wash-cycle simulations provide quantification
and further insight to the understanding of whether C. difficile spores survive the thermal and chemo-thermal conditions that can be applied by laundries. These findings verify the research by
Joanna Tarrant et al
Hellickson and Owens,8 who demonstrated the unquantified presence of C. difficile spores on inoculated and previously sterile swatches washed in a simulated healthcare wash cycle. The pre- sent study goes further in establishing that in the simulation conditions described, the use of detergent significantly reduces the number of viable spores recovered by 6–7 log10 on inoculated swatches and reduces cross-contamination by >2.5 log10 on previously sterile swatches. The 0.1–1 log10 cfu/25cm2 that may remain on swatches after a simulated healthcare wash cycle was a conservative estimate due to the inability to recover 100% of viable spores present with the vortexing method used (Fig. 1). More recently, Mackay et al9 compared washing ambulance
uniform swatches artificially contaminated with C. difficile spores at 30°C and 60°C in a Miele PW6055 (commercial) machine (Miele, Gütersloh, Germany) with Cleanline biological washing powder (Bunzl Cleaning and Hygiene Supplies, Berkshire, UK). Viable C. difficile spores were recovered from all swatches washed at 30°C and 60°C, with a range of 1.6–6.73 log10 cfu C. difficile spores remaining per swatch. This studyprovides the firstestimateof thelevelofC. difficilespore
contamination on a naturally contaminated sheet, which was sur- prisingly lowconsidering thepublishedestimate of spore shedding in CDI patients: 5–7log10 cfu per gramof feces.16The level of C. difficile spores present on the sheets before washing (51 cfu/25cm2)was comparable to that found after washing (33 cfu/25 cm2). During the simulation, the detergent system significantly
reduced spore contamination (Fig. 2). However, when processed at the commercial healthcare laundry, industrial detergent com- bined with disinfection temperatures was unable to affect a sig- nificant reduction in spores. This finding was particularly surprising because the sheets were exposed to additional repeated high-temperature cycles, through drying and calendaring, beyond what was possible in the simulations.17,18 The low levels of C. difficile spores surviving on laundered bed
sheets may pose a transmission risk via hands touching the linen. Rusin et al19 reported that Serratia rubidea and Micrococcus luteus, transferred from 100% cotton swatches to hands from an initial inoculum of 9.7 log10 cfu were 4.40 and 6.17 log10 cfu, respectively. However, these researchers noted that the hand transfer efficiency was much higher from hard surfaces than from porous surfaces such as textiles. In conclusion, in this investigation, we quantified C. difficile
spores on naturally contaminated bed sheets before and after they were washed in a washer extractor on a commercial laundry site. The thermal disinfection conditions, described in HTM 01- 04, were inadequate to fully decontaminate linen that had been naturally contaminated with C. difficile spores. The detergent and additives had the greatest effect in reducing spore burden, whether by removal or in combination with the thermal disin- fection. The surviving spores may contribute to environmental contamination when the sheets are used for bed making in the healthcare environment. Processing infected linen in commer- cial washer extractor cycles could disseminate low levels of C. difficile spores and may be contributing to sporadic outbreaks of CDI.
Acknowledgments. The authors thank the Textile Services Association for the loaned equipment, detergent supplies, and industry-specific advice. We thank PHE Newcastle for ribotyping of isolated C. difficile and the commercial laundry and hospital that provided access to their facilities.
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