Bacterial culture methods
Clostridium difficile was grown on Tryptic Soy agar containing 5% sheep blood, vitamin K and hemin (BAK) under anaerobic conditions. All plates were incubated in an anaerobic chamber. To promote sporulation, plates inoculated with C. difficile were allowed to have prolonged incubation (usually 7 days) and then the growth was scraped off the agar surface and suspended in sterile reverse osmosis water. The organisms in the suspension were pelleted by centrifugation and washed twice with sterile reverse osmosis water. The suspension was then suspended in 70% alcohol and stored at 4°C until used. Staining by malachite green stain confirmed that the suspension was predominantly spores.
Determination of the concentration of viable spores was performed by serially diluting the spore suspension in sterile phosphate buffered saline and then inoculating 0.1 mLs of each dilution and spreading this over the surface of both BAK agar and CDMN agar (Clostridium difficile moxalactam, norfloxacin) agar (Oxoid, Mississauga, ON).
To determine how efficient the Rodac plate method was for sampling spores from surfaces, serial dilutions of C. difficile spores were prepared in ATS soil [21]. The spore preparation (0.1 mLs) was inoculated over a toilet seat surface area that was equivalent to the diameter of a Rodac plate and allowed to dry overnight. Rodac plates containing CDMN agar were then pressed onto the surface for approximately 5 seconds. The plates were incubated anaerobically for 48 hours and the colonies counted.
UV-visible marker (UVM) for cleaning assessment of toilets
The UVM used for this study was a lotion (Glitterbug® from Brevis Corp., USA). This lotion is non-toxic and water soluble so it is readily removed by cleaning with soap and water solutions. Figure 1 shows how the UVM is not readily visible under regular room lighting but is visible when exposed to short-wave UV light. A hand-held UV light was used for visualization of the marker. The UVM was applied to the underside of the toilet seat or commode and was visually inspected the following day to determine if it had been removed or not. A visual score for residual marker was used; 3 represented heavy fluorescence, 2 represented moderate fluorescence, 1 represented light fluorescence, and 0 represented no fluorescence. Using this numeric scoring system based on visual inspection, an average cleaning score could be determined. Initial testing confirmed that if no cleaning was performed then the UVM showed heavy fluorescence that lasted for at least 7 days after it was inoculated.
Culture for C. difficilefrom patient-used toilets
Rodac plates containing CDMN agar were used to sample the commodes or toilets in patient rooms. For each toilet (or commode) the agar surface of one plate was sequentially pressed onto the armrest (if present), the underside of the toilet lid (if present), the toilet seat surface, the toilet seat underside, and the inside rim of the upper portion of the toilet bowl. These are all surfaces that should be cleaned as part of regular toilet cleaning. The Rodac plates were then placed into an anaerobic jar and incubated for 48 hours under anaerobic conditions. Suspect colonies were sub cultured to obtain pure cultures and then were confirmed as C. difficile based on colony morphology, Gram stain, colony fluorescence under UV light and agglutination using C. difficile latex (Microgen Bioproducts, Surrey, UK). The isolates were confirmed as being toxigenic by growing the isolate in Fastidious Anaerobe Broth (FAB) (Lab M Limited, Bury, U.K.) and testing the culture supernatant for Toxin B using the Bartel's CPE assay (Carlsbad, CA). All C. difficile isolates were stored as frozen stocks in skim milk at -70°C.
Housekeeping standard cleaning protocol
Once daily the toilets (and all other high-contact areas in the bathroom) were cleaned and disinfected using PerDiem® (3% Stabilized Hydrogen peroxide from Virox, Mississauga, Canada) at a 1:256 use-dilution (i.e. a final concentration of 0.01% (w/v) stabilized hydrogen peroxide (SHP)). PerDiem® contains 3% (w/v) stabilized hydrogen peroxide as the active agent as well as proprietary "builders and stabilizers." The cleaning protocol consisted of spritzing the SHP solution to wet all of the surfaces of the toilet (or commode) and allowing this to contact the surface while other parts of the bathroom were cleaned. The housekeeping instructions indicate the SHP solution should be allowed to dwell for 10 minutes prior to wiping it off; however, observational assessment of actual practice indicated that the contact time was about three minutes. After about three minutes the toilet was wiped with a cloth rag that had been wet with the same cleaning agent. The cleaning rags were used for one patient toilet only and then were sent for laundering.
Housekeeping protocol for patients on CDAD Isolation
The cleaning process was the same as indicated above however, each room (including the toilet) was cleaned twice daily (morning and afternoon) and the use-dilution for the PerDiem® was 1:64 (i.e. final concentration of 0.05% SHP).
Study enrolment
The objective was to compare compliance of housekeepers with the cleaning protocol for toilets in isolation and non-isolation rooms. Ethics and research approval for this study was obtained. Patients were enrolled in one of the following arms of the study:
Arm 1
Patients enrolled in Arm 1 of the study had diarrhea, laboratory confirmed CDAD, and were on isolation precautions. The toilets used by these patients were inoculated with UVM each weekday and then visually inspected the next day to determine if the UVM had been removed. Toilets were also sampled each weekday for the presence of detectable C. difficile spores using Rodac plates containing CDMN agar. The use-dilution of the 3% SHP cleaning agent was 1:64 (0.05% final SHP concentration) and cleaning was performed twice per day (as per the facility's housekeeping policy). If commodes were used by the patient they were also monitored using UVM and Rodac plates. There may be up to four patients sharing the same toilet facilities.
Arm 2
Patients enrolled in Arm 2 of the study had diarrhea, laboratory confirmation that they did not have CDAD and they were not on isolation precautions. The toilets used by these patients were inoculated with UVM each weekday and then visually inspected the next day to determine if the UVM had been removed. Toilets were also sampled each weekday for the presence of detectable C. difficile spores using Rodac plates containing CDMN agar. The use-dilution of the 3% SHP cleaning agent was 1:256 (0.01% final SHP concentration), and toilet cleaning was performed once per day (as per the facility's housekeeping policy). If commodes were used by the patient they were also monitored using UVM and Rodac plates. There may be up to four patients sharing the same toilet facilities.
Routine Ward cleaning
In addition to the monitoring of toilets as outlined in Arms 1 and 2, routine ward cleaning was assessed by prospectively using UVM to monitor the toilets of all rooms on three separate wards on a daily basis over a 1 week period (Monday to Friday). All toilets were included regardless of whether the patients in the room had diarrhea or not. The toilets should have been cleaned once each day using PerDiem® the SHP cleaning agent at 0.01% final concentration.
As required by the ethics review committee, all housekeeping staff was informed about the study and the use of UVM as a measure of cleaning compliance. However, they did not know which patient rooms would be involved. The results of the marker were not traced back to individual housekeepers and punitive action was not taken even if residual marker was detected.