Activity of a dry mist hydrogen peroxide system against environmental Clostridium difficile contamination in elderly care wards

Clostridium difficile causes serious healthcare-associated infections. Infection control is difficult, due in part to environmental contamination with C. difficile spores. These spores are relatively resistant to cleaning and disinfection. The activity of a dry mist hydrogen peroxide decontamination system (Sterinis((R))) against environmental C. difficile contamination was assessed in three elderly care wards. Initial sampling for C. difficile was performed in 16 rooms across a variety of wards and specialties, using Brazier's CCEY (cycloserine-cefoxitin-egg yolk) agar. Ten rooms for elderly patients (eight isolation and two sluice rooms) were then resampled following dry mist hydrogen peroxide decontamination. Representative isolates of C. difficile were typed by polymerase chain reaction ribotyping. C. difficile was recovered from 3%, 11% and 26% of samples from low, medium and high risk rooms, respectively. In 10 high risk elderly care rooms, 24% (48/203) of samples were positive for C. difficile, with a mean of 6.8 colony-forming units (cfu) per 10 samples prior to hydrogen peroxide decontamination. Ribotyping identified the presence of the three main UK epidemic strains (ribotypes 001, 027 and 106) and four rooms contained mixed strains. After a single cycle of hydrogen peroxide decontamination, only 3% (7/203) of samples were positive (P<0.001), with a mean of 0.4 cfu per 10 samples ( approximately 94% reduction). The Sterinis((R)) hydrogen peroxide system significantly reduced the extent of environmental contamination with C. difficile in these elderly care rooms. This relatively quick and user-friendly technology might be a more reliable method of terminally disinfecting isolation rooms, following detergent cleaning, compared to the manual application of other disinfectants.     

Activity of three disinfectants and acidified nitrite against Clostridium difficile spores.

OBJECTIVE: To identify environmentally safe, rapidly acting agents for killing spores of Clostridium difficile in the hospital environment. DESIGN: Three classic disinfectants (2% glutaraldehyde, 1.6% peracetyl ions, and 70% isopropanol) and acidified nitrite were compared for activity against C. difficile spores. Four strains of C. difficile belonging to different serogroups were tested using a dilution-neutralization method according to preliminary European Standard prEN 14347. For peracetyl ions and acidified nitrite, the subjective cleaning effect and the sporicidal activity was also tested in the presence of organic load. RESULTS: Peracetyl ions were highly sporicidal and yielded a minimum 4 log10 reduction of germinating spores already at short exposure times, independent of organic load conditions. Isopropanol 70% showed low or no inactivation at all exposure times, whereas glutaraldehyde and acidified nitrite each resulted in an increasing inactivation factor (IF) over time, from an IF greater than 1.4 at 5 minutes of exposure time to greater than 4.1 at 30 minutes. Soiling conditions did not influence the effect of acidified nitrite. There was no difference in the IF among the 4 strains tested for any of the investigated agents. Acidified nitrite demonstrated a good subjective cleaning effect and peracetyl ions demonstrated a satisfactory effect. CONCLUSIONS: Cidal activity was shown against C. difficile spores by glutaraldehyde, peracetyl ions, and acidified nitrite. As acidified nitrite and peracetyl ions are considered to be environmentally safe chemicals, these agents seem well suited for the disinfection of C. difficile spores in the hospital environment.     

Activity in vitro of hydrogen peroxide vapour against Clostridium difficile spores

Clostridium difficile spores are shed in high numbers by infected patients and are resistant to desiccation and some disinfectants. We explored the in?vitro activity of hydrogen peroxide vapour (HPV) against several strains of C.?difficile spores using a spore-carrier test. Spores were dried on polyvinyl chloride or laminate carriers at mean concentrations of 4.7-6.9 log(10) spores/carrier, which were then decontaminated using HPV. C.?difficile was completely eradicated from the exposed carriers regardless of the C.?difficile strain or surface used. HPV can be considered for the eradication of C.?difficile spores from the hospital environment.     

Molecular epidemiology of endemic Clostridium difficile infection

This is the first study to provide a comprehensive insight into the molecular epidemiology of endemic Clostridium difficile and particularly that associated with a recently recognized epidemic strain. We DNA fingerprinted all C. difficile isolates from the stools of patients with symptomatic antibiotic-associated diarrhoea and from repeated samples of the inanimate ward environment on two elderly medicine hospital wards over a 22-month period. Notably, C. difficile was not recoverable from either ward immediately before opening, but was found on both wards within 1-3 weeks of opening, and the level of environmental contamination rose markedly during the first 6 months of the study period. C. difficile infection (CDI) incidence data correlated significantly with the prevalence of environmental C. difficile on ward B (r = 0.76, P < 0.05) but not on ward A (r = 0.26, P > 0.05). We found that RAPD and RS-PCR typing had similar discriminatory power, although, despite fingerprinting over 200 C. difficile isolates, we identified only six distinct types. Only two distinct C. difficile strains were identified as causing both patient infection and ward contamination. Attempts to determine whether infected patients or contaminated environments are the prime source for cross-infection by C. difficile had limited success, as over 90% of C. difficile isolates were the UK epidemic clone. However, a non-epidemic strain caused a cluster of six cases of CDI, but was only isolated from the environment after the sixth patient became symptomatic. The initial absence of this strain from the environment implies patient-to-patient and/or staff-to-patient spread. In general, routine cleaning with detergent was unsuccessful at removing C. difficile from the environment. Understanding the epidemiology and virulence of prevalent strains is important if CDI is to be successfully controlled.     

An investigation into techniques for cleaning mold-contaminated home contents

This study examined the efficacy of the following treatments to reduce selected fungal spore and mycotoxin levels on materials commonly found in home contents: (1) gamma irradiation at a 10-13 kiloGray exposure, (2) a detergent/bleach wash, and (3) a steam cleaning technique. A minimum of six replicates were performed per treatment. Paper, cloth, wood, and carpet were inoculated with either fungal spores (Stachybotrys chartarum, Aspergillus niger, Penicillium chrysogenum, or Chaetomium globosum) at 240,000 spores/2.54 cm2 of material or with the mycotoxins roridin A, T-2, and verrucarin A at 10 microg per 2.54 cm2 of material. Treatments were evaluated with an agar plating technique for fungal spores and a yeast toxicity culture assay for mycotoxins. Results showed that gamma irradiation inactivated fungal spores, but the treatment was not successful in inactivating mycotoxins. The washing technique completely inactivated or removed spores on all materials except for C. globosum, which was reduced on all items except paper (p < 0.05). Washing inactivated all mycotoxins on paper and cloth but not on carpet or untreated wood (p < 0.001). The steam cleaning treatment did not completely eliminate any fungal spores; however, it reduced P. chrysogenum numbers on all materials, C. globosum was reduced on wood and carpet, and S. chartarum was reduced on wood (p < 0.05). Steam cleaning was unsuccessful in inactivating any of the tested mycotoxins. These results show that the bleach/detergent washing technique was more effective overall in reducing spore and mycotoxin levels than gamma irradiation or steam cleaning. However, the other examined techniques were successful in varying degrees.     

Cleaning of blood-contaminated reprocessed angiographic catheters and spinal needles.

OBJECTIVES: To evaluate the efficacy of a multistep cleaning method using a cleaner and a chemical disinfectant on blood-contaminated angiographic catheters and spinal needles intended to be sterilized by hydrogen peroxide gas plasma. METHOD: A mixture of radiopaque iodine contrast, bovine blood (plus ethylenediaminetetraacetic acid), and a suspension of Bacillus subtilis spores was used to simulate catheterization and needle use. The mixture was a 1:1 proportion of contrast and blood, inoculated so that there was a final concentration of B subtilis spores of 1.0x10(6) colony-forming units (CFU)/mL. The inoculated devices were cleaned using a hydrogen peroxide solution at a concentration of 1.5+/-0.5 percent by weight, followed by distilled water with enzymatic detergent. After drying, the devices were sterilized with hydrogen peroxide gas plasma. RESULTS: The initial B subtilis spore concentration inoculated into catheters and needles varied from 2.12x10(4) to 2.74x10(7) CFU/mL. The residual load of B. subtilis spores after cleaning varied from zero (no count) to a maximum of 200 CFU/device. The multistep cleaning procedure was responsible for an average 5log10 reduction of B. subtilis spores in the catheter and needle lumens. CONCLUSIONS: The hydrogen peroxide and enzymatic detergent aqueous solutions were shown to be efficacious when used as part of a multistep cleaning method. The low level of microbial contamination prior to sterilization with hydrogen peroxide gas plasma assured that the intended sterility assurance level was reached.     

Prospective evaluation of environmental contamination by Clostridium difficile in isolation side rooms.

We determined prospectively the frequency, persistence and molecular epidemiology of Clostridium difficile environmental contamination after detergent-based cleaning in side rooms used to isolate patients with C. difficile diarrhoea. Approximately one-quarter of all environmental sites in side rooms sampled over four-week periods were contaminated with C. difficile. The overall side room prevalence of environmental C. difficile declined from 35% initially, to 24% in week 2, 18% in week 3, and 16% in week 4. The bed frame was the most common site from which C. difficile was recovered, although the floor was the most contaminated site in terms of total numbers of colonies. C. difficile was recovered significantly more frequently from swabs plated directly on to C. difficile selective media containing lysozyme than from enrichment broth (P< 0.001), emphasizing the benefit of lysozyme supplementation. The great majority of C. difficile isolates (87% of all isolates, 84% of patient isolates) was indistinguishable from the UK epidemic strain (PCR ribotype 1). It thus could not be determined whether environmental contamination was a cause or a consequence of diarrhoea. Our findings highlight the need for improved approaches to hospital environmental hygiene, and call into question current UK guidelines that recommend detergent-based cleaning to remove environmental C. difficile. In particular, improved cleaning of frequently touched sites in the immediate bed space area is required.     

Value of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery of Clostridium difficile

Clostridium difficile is an increasingly prevalent nosocomial pathogen. Environmental contamination by spores is believed to be a major factor propagating the spread of C. difficile. Various approaches including the use of bile salts have been described to enhance the recovery of C. difficile from clinical and environmental specimens. We found that lysozyme (5 mg/L) incorporated into a selective medium containing bile salts significantly increased the recovery of C. difficile from swabs of 197 environmental sites (11% versus 24% samples positive, P< 0.01). Furthermore, in a separate series of experiments additional use of cooked meat broth enrichment significantly enhanced the recovery of C. difficile (35% versus 45%, P = 0.009). Conversely, we found that pre-exposure to alkaline thioglycollate did not improve the yield of C. difficile. Lysozyme incorporation markedly increases the recovery of C. difficile from environmental samples probably by stimulation of spore germination. Our findings suggest that previous attempts to determine the level of environmental C. difficile contamination have markedly underestimated the true prevalence of this pathogen.     

Survival of Clostridium difficile on copper and steel: futuristic options for hospital hygiene

Clostridium difficile is rapidly becoming a major cause of hospital-acquired infections worldwide, due in part to transmission of the faecal pathogen between contaminated hands and contact surfaces. Accordingly, this study evaluated survival of C. difficile vegetative cells and spores on the contact surface commonly found in healthcare settings, stainless steel, compared to five copper alloys (65-100% copper content). C. difficile requires prolonged incubation to grow and therefore the total number and number of viable cells was estimated using a fluorescence dual-staining technique. For viability assessment the redox dye 5-cyano-2,3-ditolyl tetrazolium (CTC) was used to measure metabolic activity. Results demonstrated that copper alloys with a copper content >70% provide a significant reduction in survival of C. difficile vegetative cells and spores on copper alloys compared with stainless steel. Complete death of spores was observed after 24-48 h on copper alloys whereas no significant death rate was observed on stainless steel even after 168 h. The use of CTC gave comparable results to culture and offers a more rapid viability analysis (8 h) than culture. The results suggest that using copper alloys in hospitals and other healthcare facilities could offer the potential to reduce spread of C. difficile from contaminated surfaces.     

Gaseous and air decontamination technologies for Clostridium difficile in the healthcare environment

The recent data for hospital-acquired infections suggest that infection rates for meticillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile are beginning to decrease. However, while there is still pressure to maintain this trend, the resistance of C. difficile spores to standard detergents continues to present a problem for many UK hospitals trying to prevent its spread or control outbreaks. Alternative disinfection technologies such as gaseous decontamination are currently being marketed to the healthcare sector as an alternative/supplement to manual disinfection, and have been shown to be effective in reducing environmental contamination. When used correctly, they offer a complementary technology to manual cleaning that increases the probability of an effective reduction in viability and provides a comparatively uniform distribution of disinfectant. Three gaseous decontamination technologies are examined for their suitability in reducing environmental contamination with C. difficile: gaseous hydrogen peroxide, chlorine dioxide and ozone. Air decontamination and UV-based technologies are also briefly described. We conclude that while there is a role to play for these new technologies in the decontamination of ward surfaces contaminated with C. difficile, the requirement for both a preclean before use and the limited 'in vivo' evidence means that extensive field trials are necessary to determine their cost-effectiveness in a healthcare setting.     

The presterilization microbial load on used medical devices and the effectiveness of hydrogen peroxide gas plasma against Bacillus subtilis spores.

OBJECTIVES: To determine the microbial load found on used critical medical devices (5 spinal anesthesia needles, 21 catheters, and 28 sheaths) prior to sterilization and to evaluate the effectiveness of hydrogen peroxide gas plasma against inoculated Bacillus subtilis var globigii (American Type Culture Collection 9372) spores. METHODS: Membrane filter and pour-plate methods were applied to estimate total microbial loads (aerobic and anaerobic, mesophilic and thermophilic, vegetative and spore forms). Spinal anesthesia needles (102 units) and sheath components (61 units) were inoculated with a suspension of B. subtilis spores. After drying, the devices were sterilized with hydrogen peroxide gas plasma. RESULTS: Higher counts of aerobic, mesophilic, and fungal organisms were recovered when the drying period was insufficient. Anaerobic spores were not found in any analyzed presterilization items. The hydrogen peroxide gas plasma effected a 5 to 7 log10-fold reduction in B. subtilis spore counts in well-dried needles and sheath components. CONCLUSIONS: The success of hydrogen peroxide gas plasma sterilization depends mostly on educating the staff to assure well-cleaned and dried reusable medical devices, allowing penetration of the hydrogen peroxide gas plasma into the critical points of the items and providing a reduction in organisms.     

Epidemic Clostridium difficile Ribotype 027 in Chile

TO THE EDITOR: The increased severity of Clostridium difficile infection is primarily attributed to the appearance of an epidemic strain characterized as PCR ribotype 027 (1). The only report that identified epidemic C. difficile ribotype 027 in an American country outside of North America comes from Costa Rica, raising the possibility that strains 027 might also be present in other countries of Latin America (2). Several studies between 2001 and 2009 have been conducted in South American countries to detect the incidence of C. difficile infection in hospitalized patients, but they did not identify which C. difficile strains were causing these infections (3).     

Comparison of the effect of detergent versus hypochlorite cleaning on environmental contamination and incidence of Clostridium difficile infection

To determine how best to decontaminate the hospital environment of Clostridium difficile, we carried out a cross-over study on two elderly medicine wards to determine whether cleaning with a hypochlorite disinfectant was better than using neutral detergent in reducing the incidence of C. difficile infection (CDI). We examined 1128 environmental samples in two years, 35% of which grew C. difficile. There was a significant decrease of CDI incidence on ward X, from 8.9 to 5.3 cases per 100 admissions (P<0.05) using hypochlorite, but there was no significant effect on ward Y. On ward X the incidence of CDI was significantly associated with the proportion of culture-positive environmental sites (P<0.05). On ward Y the only significant correlation between CDI and C. difficile culture-positive environmental sites was in patient side-rooms (r=0.41, P<0.05). The total daily defined doses of cefotaxime, cephradine and aminopenicillins were similar throughout the trial. These results provide some evidence that use of hypochlorite for environmental cleaning may significantly reduce incidence of CDI, but emphasize the potential for confounding factors.     

Airborne hydrogen peroxide for disinfection of the hospital environment and infection control: a systematic review

We reviewed the effectiveness of airborne hydrogen peroxide as an environmental disinfectant and infection control measure in clinical settings. Systematic review identified ten studies as eligible for inclusion. Hydrogen peroxide was delivered in the form of vapour and dry mist in seven and three studies, respectively. Pathogens evaluated included meticillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile and multiple bacterial types, in five, three, and two studies, respectively. Before the application of any cleaning intervention, 187/480 (39.0%; range: 18.9-81.0%) of all sampled environmental sites were found to be contaminated by the studied pathogens in nine studies that reported specific relevant data. After application of terminal cleaning and airborne hydrogen peroxide, 178/630 (28.3%; range: 11.9-66.1%) of the sampled sites in six studies and 15/682 (2.2%; range: 0-4.0%) of the sampled sites in ten studies, respectively, remained contaminated. Four studies evaluated the use of hydrogen peroxide vapour for infection control. This was associated with control of a nosocomial outbreak in two studies, eradication of persistent environmental contamination with MRSA and decrease in C. difficile infection in each of the remaining two studies.     

The effect of Perasafe and sodium dichloroisocyanurate (NaDCC) against spores of Clostridium difficile and Bacillus atrophaeus on stainless steel and polyvinyl chloride surfaces

Clostridium difficile is an important cause of nosocomial diarrhoea. The aim of this study was to evaluate the potential for Perasafe, a recently introduced biocide, to contribute to control of C. difficile spores in the patient environment, in comparison with the chlorine-releasing agent sodium dichloroisocyanurate (NaDCC). These agents were evaluated against a water control, in a surface test on stainless steel and polyvinyl chloride (PVC) floor covering, materials commonly found in the hospital environment. The organisms studied were a toxigenic clinical isolate of C. difficile, and Bacillus atrophaeus (formerly B. subtilis var niger). The data indicate that in our in vitro system, Perasafe was significantly more active than NaDCC (1000 ppm available chlorine) against C. difficile spores dried on stainless steel surfaces, and against B. atrophaeus on PVC floor covering material, achieving mean log10 reduction factors in viable counts of 6 and 5.5, respectively, at 10 min exposures. Perasafe appeared to be less lethal in 10 min exposures to C. difficile spores fixed on PVC floor covering material. In general, 1000 ppm chlorine generated from NaDCC showed lower log10 reduction factors in viable counts at 10 min, ranging from 0.7 to 1.5, than Perasafe which ranged from 2.7 to 6.0. The potential efficacy of Perasafe in reducing the density of C. difficile spores in the patient environment in hospitals, nursing homes or other long-stay facilities should be evaluated in field studies.     

Lack of enhanced effect of a chlorine dioxide-based cleaning regimen on environmental contamination with Clostridium difficile spores

Spores of Clostridium difficile may play a significant role in transmission of disease within the healthcare environment and are resistant to a variety of detergents and cleaning fluids. A range of environmental cleaning agents has recently become available, many of which claim to be sporicidal. We investigated the effect of changing to a chlorine dioxide-based cleaning regimen on C.?difficile environmental contamination and patient infection rates. The prevalence of environmental contamination was unaffected with a rate of 8% (9/120) before and 8% (17/212) following the change. Rates of patient infection were also unchanged during these periods.     

Evaluation of microbicidal activity of a new disinfectant: Sterilox 2500 against Clostridium difficile spores, Helicobacter pylori, vancomycin resistant Enterococcus species, Candida albicans and several Mycobacterium species

The microbicidal activity of a new disinfectant Sterilox, a super-oxidized water, containing a mixture of oxidizing substances, was tested against Clostridium difficile spores, Helicobacter pylori, vancomycin resistant Enterococcus species, Candida albicans and several Mycobacterium species using membrane filters. All tests were performed in duplicate with and without added horse serum at 1% and 5% v/v. Distilled water, 0.35% peracetic acid (Nu-Cidex) and 2% glutaraldehyde were included as controls. Sterilox: spore suspension (9:1 v/v) achieved log10 kill of > 5 with 5% horse serum in 2 min against H. pylori, vancomycin resistant Enterococcus species, C. albicans and four atypical Mycobacterium species: M. avium, M. chelonei, M. xenopi and M. smegmatis. Sporicidal activity of Sterilox against Clostridium difficile was markedly diminished in the presence of 5% horse serum. Sterilox may be an effective alternative in endoscopy units, as it is a potent microbicidal agent and the manufacturer claims it is not corrosive to metal and is nontoxic to biological tissues.     

Efficacy of low-concentration iodophors for germicidal hand washing

The efficacy of iodophor germicides containing different concentrations of available iodine against transient (inoculated) bacteria and the natural hand microflora was compared with chlorhexidine gluconate (2 and 4%) liquid detergent (Hibitane), non-germicidal soap and a tap water rinse. The tap water rinse was ineffective compared with all other treatments. Only 4% chlorhexidine gluconate liquid detergent and iodophor containing 0.75% available iodine were significantly better than the non-germicidal soap for reduction of transient bacteria, Escherichia coli and Pseudomonas fluorescens, that had been inoculated onto hands. These agents also caused a significant reduction in the number of 'natural' microorganisms released from hands after a standard 15 s hand wash. The low-concentration iodophor products and the product containing 2% chlorhexidine gluconate failed to give results significantly better than the non-germicidal control soap. Baird-Parker medium and standard aerobic plate counts were highly correlated (r = 0.82), so that for studies of Gram-negative bacteria inoculated onto hands as a transient microflora, counts on Baird-Parker medium give a reasonable indication of the natural (residual) hand microflora.     

Evaluation of the sporicidal activity of different chemical disinfectants used in hospitals against Clostridium difficile

Decontamination of surfaces and medical equipment is integral to the control of Clostridium difficile transmission, and many products claim to inactivate this bacterium effectively. Thirty-two disinfectants were tested against spores of C. difficile in a suspension test based on European Standard BS EN 13704:2002, with contact times of 1 and 60 min in simulations of clean (0.3% albumin) and dirty (3% albumin) conditions. The addition of a 1-min contact time was chosen as a more realistic simulation of probable real-life exposures in the situation being modelled than the 60 min specified by the Standard. The manufacturer's lowest recommended concentrations for use were tested. Sixteen products achieved >10(3) reduction in viability after 60 min (the pass criterion for the Standard) under both clean and dirty conditions. However, only eight products achieved >10(3) reduction in viability within 1 min under dirty conditions. Three products failed to reduce the viability of the C. difficile spores by a factor of 10(3) in any of the test conditions. This study highlights that the application of disinfectants claiming to be sporicidal is not, in itself, a panacea in the environmental control of C. difficile, but that carefully chosen environmental disinfectants could form part of a wider raft of control measures that include a range of selected cleaning strategies.     

Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery

Bacillus subtilis has been successfully engineered to express heterologous antigens genetically fused to surface-exposed spore coat proteins as a vaccine vehicle endowed with remarkable heat resistance and probiotic effects for both humans and animals. Nonetheless, the immunogenicity of passenger antigens expressed by B. subtilis spores is low particularly following oral delivery. In this work, we describe a new episomal expression system promoting enhanced immunogenicity of heterologous antigens carried by B. subtilis strains, either in the form of spores or vegetative cells, following oral or parenteral delivery to mice. Based on a bi-directional replicating multicopy plasmid, the gene encoding the B subunit of the heat-labile toxin (LTB), produced by enterotoxigenic Escherichia coli (ETEC) strains, was cloned under the control of the B. subtilis glucose starvation inducible (gsiB) gene promoter, active in vegetative cells submitted to heat and other stress conditions. The recombinant plasmid proved to be structurally and segregationally stable in both cells and spores under in vitro and in vivo conditions. Moreover, BALB/c mice orally immunized with B. subtilis cells or spores elicited enhanced anti-LTB systemic (serum IgG) and secreted (fecal IgA) antibody responses, thus, suggesting that antigen expression occurred during in vivo transit. These results indicate that the new episomal expression system may improve the performance of B. subtilis as a live orally-delivered vaccine carrier.