Environmental Sampling for Clostridium difficile on Alcohol-Based Hand Rub Dispensers in an Academic Medical Center

Abstract

Background:

Clostridum difficile is a gram-positive, spore-forming anaerobic bacillus that has substantial associated morbidity, mortality, and associated healthcare burdens. Clostridium difficile spores are not destroyed by alcohol. Alcohol gel dispensers are used commonly as the hand sanitization method of choice in hospitals. It is possible that gel dispensers are fomites for C. difficile.

Methods:

Thirty alcohol-based gel dispenser handles outside of rooms of patients with active C. difficile infection were sampled. The samples were assessed for C. difficile by both culture and polymerase chain reaction (PCR). The samples were also assessed for other organisms by culture.

Results:

No C. difficile was cultured or detected by PCR on any of the gel dispensers. Coagulase-negative Staphyloccus spp., diptheroids, and Bacillus spp. were the organisms detected most commonly.

Conclusion:

At our institution, C. difficile is not present on alcohol-based gel dispensers, but other potentially pathogenis are.

C lostridum difficle is a gram-positive, spore-forming anaerobic bacillus. It is a major nosocomial pathogen responsible for 15–25% of antibiotic-associated diarrhea and nearly all cases of pseudomembranous colitis [1,2]. Well established risk factors for C. difficile infection (CDI) include recipients of antibiotic therapy, age older than 65 years, associated medical co-morbidities, immunosuppression, proton pump inhibitors, and length of hospital stay [3]. At the turn of the century, a highly virulent strain of C. difficile emerged, characterized as ribotype 027/NAP1. This strain typically causes more severe infections that include septic shock, toxic megacolon, perforation, and death. Whereas the attributable mortality of typical C. difficile has typically been estimated to be ∼2%, the attributable mortality of ribotype 027 was found to be 6.9% [4]. Emergence of this strain was concomitant with the general increase in incidence of CDI in North America and Northern Europe [5 –7]. Epidemic clones such as ribotype 027 have a better sporulation capacity than other C. difficile strains [8]. The persistence of C. difficile spores in the environment creates a reservoir from which new epidemics can arise. Approximately 20% of adults will become colonized with C. difficile during an inpatient hospital stay [9]. Clostridium difficile has a large financial impact on the U.S. healthcare system. Patient-incurred adjusted cost for C. difficile-associated diarrhea has been estimated to cost between $2,000–8,000 for index cases, and $13,000–$18,000 for recurrent cases [10,11]. Clostridium difficile infections now account for approximately $3 billion per year in health care expenditure in the U.S. [4,12]. Preventing the transmission of C.difficile can have meaningful impacts on patient care as well as cost savings.

Clostridium difficile is spread by the oral-fecal route via spores, and the hands of health care workers are common vectors. Rooms that have patients with CD are contaminated commonly with both C. difficile in its vegetative state and C. difficile spores [3,13]. Environmental contamination is more common when patients are actively infected, providing further evidence of oral-fecal spread and the necessity of hand hygiene and barrier precautions [3].

Alcohol-containing gel dispensers have been utilized to improve hand hygiene both in the intensive care unit (ICU) and hospital wards. This practice was defined by the 2002 World Health Organization Guideline for Hand Hygiene in Health Care Settings, which advocates alcohol-based hand rubs (ABHRs) before and after patient contact [14]. Whereas ABHR dispensers are active against many nosocomial pathogens, their role in the setting of CDI is less clear. Alcohol-based hand rubs are ineffective at destroying C. difficile spores [14 –17] being no more effective at removing spores than water. Non-pathogenic strains of C. difficile spores are transmitted 30% of the time after a single handshake after using AHBRs [15]. Clostridium difficile is a common environmental pathogen and CDI is limited to strains able to replicate in the human host as well as to produce toxin [18]. Historically, environmental studies evaluating the presence of C. difficile have utilized culture followed by a toxin production assay for confirming the presence of the toxigenic organism [19 –24]. However, several of these studies were performed prior to the advent of rapid polymerase chain reaction (PCR) techniques. In clinical specimens, multiple studies have demonstrated nearly equivalent sensitivities and specificities of PCR to culture for detecting organisms from stool samples [18]. However, no attempt has been made to our knowledge to compare PCR to culture for evaluating environmental samples for C. difficile.

We hypothesized that there would be some measurable percentage of C. difficile spores on ABHR dispensers in the hospital. We conducted a pilot study with two specific aims. The first aim was to determine if ABHR were contaminated with C. difficile spores. To explore this aim, we studied the dispensers with the highest likelihood of contamination, (i.e., dispensers outside rooms of patient's with active C. difficile infection). We also sought to validate PCR for environmental sampling against the gold standard of culture.

Patients and Methods

The electronic medical record was sampled daily by the infection control team for patients with active CDI. Active CDI was defined as patients with more than three loose bowel movements per day and a positive PCR stool assay for toxin B gene. We evaluated alcohol dispensers outside rooms of patients ≥18 years of age at a quaternary academic medical center. Sampling of the dispensers was conducted using methodology that has been validated previously [25]. Briefly, a sterile BBL CultureSwab™ (2 applicator tips) was moistened with sterile normal saline (Beckson Dickinson, Franklin Lakes, NJ). The swabs were then rubbed over the dispenser paddle five times ensuring that both swabs, and both sides of each swab were exposed to the dispenser paddle. All dispensers were EcoLab^® paddle-based dispensers (EcoLab, St. Paul, MN). One of the swabs was then run on an Xpert C. difficile/Epi cartridge in the Cepheid GeneXpert™ four-chamber PCR analyzer according the manufacturer's instructions (Cepheid, Sunnyvale, CA). The swab tip was removed and placed in the manufacturer's extraction solution and then underwent vortex for 10 sec. The extraction solution was then placed into the Xpert C. difficile/Epi cartridge, which was then placed in the Cepheid GeneXpert instrument. The extraction process was performed with barrier precautions. Polymerase chain reaction was run for both Toxin B and ribotype 027 with the manufacturer's internal control.

The second swab was placed in a chopped meat carbohydrate (CCM) test tube (Anaerobe Systems, Morgan Hill, CA). The tip of the swab was twirled for 15 sec and then was pressed against the side of the test tube. It was then incubated for 48 h and assessed for turbidity. Samples were gram stained and plated onto cycloserine cefoxitin fructose agar with horse blood and taurocholate (CCFA-HT) (Anaerobe Systems, Morgan Hill, CA), BBL (Beckson Dickinson, Franklin Lakes, NJ), and Blood Agar Plate (BAP) (Anaerobe Systems, Morgan Hill, CA). The plates were then incubated at 37C for 48 h in the anaerobic chamber. The CCFA-HT plates were screened after 48 h for flat, gray, shiny colonies with spreading edges. If plates had appropriate morphology, they were tested with the RapID ANA system (Thermo Fisher Scientific, Waltham, MA), evaluated for horse manure smell, and UV fluorescence, and then be tested for toxin B and the ribotype 027 genotype using the Cepheid Cdiff/Epi PCR system. If no turbid growth was noted after 48 h, the cultures were incubated an additional 72 h for further growth then only plated on the CCFA-HT plates to see if growth was present but at low levels. A licensed clinical laboratory technician blinded to the PCR results evaluated the CCFA-HT, BBL, and BAP plates. Any samples returning as positive for C.difficile either by culture or PCR were immediately reported to Infection Control. Informed consent was determined by the institutional review board to not be required for this study.

Results

Thirty alcohol-based gel dispensers outside of 30 separate rooms, housing patients individually with CDI were sampled during a three-week time period. The gel dispensers were sampled at random intervals throughout the 24-h day. Gel dispensers outside of both ICU and ward rooms were sampled. None of the dispensers tested positive for C. difficile either by culture or by PCR. Even after 120 h, there was no C. difficile growth on any of the CCFA-HT plates. However, several other species were cultured (Table 1). No C. difficile was either cultured or found with PCR, so sensitivity and specificity were unable to be assessed.

Table 1.

Bacteria Isolated from Levers of Alcohol-Based Gel Dispensers

SAMPLE	GRAM STAIN	BAP	BRUCELLA	CCFHT
1	GPC	CNS	CNS	-
2	GPC	CNS	Mixed CNS x2	-
3	Mixed GPB and GPC	CNS+Diptheroid	Mixed CNS and Diptheroid	-
4	GPB	Bacillus spp.	Bacillus spp.	-
5	GPC	CNS	CNSx2	-
6	GPC in groups	CNS	CNS	-
7	Pleomorphic GPB	Diptheroid	Diptheroid	-
8	GPC in groups	CNS	CNSx2	-
9	Pleomorphic GPB	Two Diptheroid	Diptheroid	-
10	-	-	-	-
11	-	-	-	-
12	Pleomorphic GPB	Diptheroid	Diptheroid	-
13	-	-	-	-
14	Pleomorphic GPB	Diptheroid	Diptheroid	-
15	Pleomorphic GPB	Diptheroid	Diptheroid	-
16	-	-	-	-
17	-	-	-	-
18	Pleomorphic GPB	Diptheroid	Diptheroid	-
19	GPB in pairs	Diptheroid	Diptheroid	-
20	Pleomorphic GPB	CNS and Diptheroid	Mixed CNS and Diptheroid	-
21	Pleomorphic GPB	Diptheroid	Diptheroid	-
22	-	-	-	-
23	-	-	-	-
24	Pleomorphic GPB	Diptheroid	Diptheroid	-
25	-	-	-	-
26	-	-	-	-
27	-	-	-	-
28	Pleomorphic GPB	Diptheroid	Two diptheroid	-
29	GPB	Bacillus spp.	Bacillus spp.	-
30	GPB	Bacillus spp.	Bacillus spp.	-

-indicates no growth.

CNS=coagulase-negative Staphylococcus spp; GPB=gram-positive bacilli; GPC=gram-positive cocci.

The predominate organism isolated from the dispensers were various diptheroids, a term used to describe pleomorphic gram-positive bacilli (n=12 (40 %)). In addition, seven coagulase-negative Staphylococcus (CNS) species and three Bacillus species were isolated. The most common finding overall was no growth.

Discussion

In this pilot study, no C. difficile colonies were identified by culture or PCR on any of the thirty alcohol-based gel dispensers sampled. Clostridium difficile spores are a well-known hospital environmental pathogen found on many surfaces in rooms with patients with active CDI [3,13]. We hypothesized that alcohol-based gel dispensers outside of these rooms, which caregivers and visitors touch, might also be contaminated with C. difficile. Hand hygiene compliance is not 100% and occasionally visitors and caregivers use the gel dispensers instead of washing with soap and water, which could transmit spores to dispensers outside of the patient rooms. However, in our pilot study evaluating these dispensers outside of rooms of patients with active CDI, no spores were found.

There are several potential explanations for this finding. First is that the environmental cleaning performed twice daily by the custodial staff was sufficient to eliminate spores from the gel dispensers. Whereas the stated cleaning goal at our institution is to clean every dispenser outside of a patient's room twice daily, it is uncertain how frequently this is achieved. This is performed with EcoLab Disinfectant 2.0™, a non-bleach-based cleanser (EcoLab, St. Paul, MN). Bleach-based cleanser is used only for cleaning in rooms of patient with C. difficile. However, it would appear that whatever frequency of cleaning is performed is sufficient to eliminate C. difficile spores on the alcohol based gel dispenser. A second possibility is that the spores were present, but in such low concentrations so as to not be picked up by culture or PCR. However, our extended culture period of 120 h should have picked up the presence of any viable spores. A third possibility is that the sampling process was not sufficient to pick up spores that were present. We consider this scenario to be less likely as our environmental sampling technique is similar to methods established previously [19,25]. Furthermore, the cultures did grow out other bacterial species.

One limitation to interpreting the findings of other bacterial species is that all cultured samples had to pass 48 h of anaerobic growth. Whereas this helped select for C. difficile, it limited secondary evaluation of other species. Only species able to tolerate anaerobic conditions would survive culture. Hence, the preponderance of coagulase-negative Staphylococcus species and diptheroids in the cultured results. The distribution of organisms that we cultured from the dispensers was different from that from the recent series Eiref et al. [25]. In their study, they found 94% of their dispensers had CNS, whereas diptheroids and Bacillus spp. only accounted for 29% of isolated organisms. In our study, diptheroids and Bacillus spp. accounted for a higher percentage of isolates and were found on 50% of our dispenser handles, whereas CNS accounted for only 23.3% of isolates. Of note, we detected no S. aureus, which Eiref et al. had cultured [25]. It is likely that the differences seen in the distribution of species reflects differences in local hospital bacterial ecology. These organisms are generally part of the normal skin flora. However, in immunosuppressed hosts they can cause disease and this highlights the importance of practicing excellent hand hygiene and barrier precautions when interacting with these patient populations.

Ultimately this pilot study calls into question the utility of putting signs above alcohol based gel dispensers outside of rooms of patients with C. difficile directing staff and visitors to handwash and not gel. Whereas this practice is not ubiquitous, it is practiced at a number of institutions, although not at the study institution. No C. difficile spores were found on any of the thirty dispensers sampled. Putting signs above gel dispensers requires additional work for the infection control team, and re-enforces non-compliance with gel dispensers. However, this does require that a strict handwashing policy is enforced and that handwashing sinks are available in or immediately outside of rooms of patients with CDI.

Conclusion

Alcohol gel dispensers located outside of rooms of patients with active CDI do not appear to be fomites for C. difficile in our institution. Coagulase-negative Staphylococci, diptheroids and Bacillus spp. were the organisms found most commonly on the gel dispenser handles. A comparison between PCR and culture was unable to be made as no positive results were obtained with either sampling method.

Footnotes

Acknowledgments

The authors thank Cepheid who provided the Cepheid Cdiff/Epi™ PCR cartridges.

Author Disclosure Statement

The authors report no conflicts of interest. The authors are responsible for the content and writing of the paper.

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