Disinfectant Wipes with Nonsporicidal Claims Transfer Clostridioides difficile Spores from Contaminated Surfaces to Uncontaminated Surfaces during the Disinfection Process

Background: Pre-wetted disinfectant wipes are increasingly being used in healthcare facilities to help address the risk of healthcare associated infections (HAI). However, HAIs are still a major problem in the US with Clostridioides difficile being the most common cause, leading to approximately 12,800 deaths annually in the US. An underexplored risk when using disinfectant wipes is that they may cross-contaminate uncontaminated surfaces during the wiping process. The objective of this study was to determine the cross-contamination risk that pre-wetted disinfectant towelettes may pose when challenged with C. difficile spores. We hypothesized that although the tested disinfectant wipes had no sporicidal claims, they will reduce spore loads. We also hypothesized that hydrogen peroxide disinfectant towelettes would present a lower cross-contamination risk than quaternary ammonium products. Methods: We evaluated the risk of cross-contamination when disinfectant wipes are challenged with C. difficile ATCC 43598 spores on Formica surfaces. A disinfectant wipe was used to wipe a Formica sheet inoculated with C. difficile. After the wiping process, we determined log10 CFU on previously uncontaminated pre-determined distances from the inoculation point and on the used wipes. Results: We found that the disinfectant wipes transferred C. difficile spores from inoculated surfaces to previously uncontaminated surfaces. We also found that wipes physically removed C. difficile spores and that hydrogen peroxide disinfectants were more sporicidal than the quaternary ammonium disinfectants. Conclusion: Regardless of the product type, all disinfectant wipes had some sporicidal effect but transferred C. difficile spores from contaminated to otherwise previously uncontaminated surfaces. Disinfectant wipes retain C. difficile spores during and after the wiping process.


Background
Pre-wetted disinfectant wipes are increasingly being used in healthcare facilities to disinfect equipment and environmental surfaces proximal to patients to help address the risk of healthcare associated infections (HAIs) [1]. This may play a significant role in reducing the incidence of certain HAIs [2,3]. However, despite efforts being made to reduce the incidence of HAIs, one out of 31 patients in the United States (US) acquires one or more HAI on a daily basis [4]. Among pathogens implicated in the incidence of HAI, Clostridioides difficile is the most common in the US [5,6]. The Center for Disease Control and Prevention (CDC) estimated that in 2017 there were approximately 223,900 hospitalized patients with C. difficile infections in the US with at least 12,800 deaths [6].
In acute care facilities, C. difficile infections result in approximately $4.8 billion in extra healthcare costs [5] due to prolonged hospital stays and readmissions [5,7].
Contamination of environmental surfaces in healthcare facilities accounts for approximately 20% of HAI [8]. Specifically, hard non-porous environmental surfaces such as bed rails [9,10,11], bedside tables [9], and call buttons [10] may harbor C. difficile spores and contribute to transmission resulting in HAI in healthcare facilities [12].
Eradication of C. difficile from environmental surfaces is particularly difficult as spores can persist on environmental surfaces for months [8,13]. Specifically, the use of nonsporicidal cleaning agents may risk increasing the sporulation rate for C. difficile [ 14]. The use of disinfectant wipes with C. difficile sporicidal claims have been a recommended strategy to reduce the incidence of HAIs [15], as wipes increase compliance with standard cleaning and disinfection practices compared to traditional "wipe and bucket" methods [16]. However, pre-wetted disinfectant wipes may pose the risk of cross-contaminating "clean" surfaces during the wiping process [17] as the standard test for the registration of towelette products rarely simulates real world wiping scenarios [3]. In 2015, Satter demonstrated that disinfectant towelettes used to wipe one-centimeter diameter disk inoculated with Staphylococcus aureus or Acinetobacter baumannii transferred about twice as much bacteria onto "clean" or otherwise not previously contaminated surfaces when compared with their bactericidal efficacy [17].
In the US, healthcare facilities commonly use visual evaluations to determine "contamination levels" on hard non-porous surfaces prior to disinfection [18].
Consequently, most disinfectant wipes are used as broad-spectrum antimicrobials on inanimate surfaces in healthcare facilities [19,20,21]. However, disinfectant wipes may also present the risk of cross-contaminating otherwise uncontaminated surfaces after a towelette has been used to disinfect a contaminated surface. In addition, the Environmental Protection Agency (EPA) has no recommendation on the maximum surface area that could be disinfected with a towelette in order to optimize bactericidal efficacy, while minimizing the risk of cross-contaminating low risk surfaces. In prior work by our group, we found that the efficacy from pre-wetted disinfectant wipes was related to the ability to keep surfaces wet for the label contact time, so "stretching" a wipe to wipe larger areas than the wipe can disinfectant may create risks of suboptimal disinfection and cross-contamination [22].
The risk of pathogen transmission by the hands of healthcare workers and patients has been widely investigated [23,24,25]. However, less work [17,26] has been done to determine the risk of cross-contamination by disinfectant wipes using real world techniques in vitro. The objective of this study was to determine the cross-contamination risk that disinfectant towelettes with no sporicidal claims may pose when challenged with C. difficile spores. We hypothesized that although the tested disinfectant wipes have no sporicidal claims, they will reduce C. difficile spore loads, but cross-contamination may still occur. On a related note, we hypothesized that towelettes with sporicidal claims will 5 present a lower cross-contamination risk than wipes without sporicidal claims. We also hypothesized that compared to quaternary ammonium products, hydrogen peroxide disinfectant towelettes will present a lower risk of cross-contaminating low risk surfaces after wiping down an area inoculated with C. difficile spores.

Disinfectants and bacterial strain used in this study
This study investigated the risk of cross-contamination of seven disinfectant towelette products; six with non-sporicidal claims and one product with sporicidal claims (Table 1).
Ready-to-use wipes containing 1.312% sodium hypochlorite with an EPA registered sporicidal claim were used as a control. C. difficile spores ATCC 43598 were produced following EPA MLB SOP-MB-28 [27] and used to study the risk of cross-contamination by disinfectant wipes following a modified version of EPA MLB SOP-MB-31 [27].

Test Surface Sterilization, Inoculation And Disinfection
A two-meter square area of Formica sheeting was marked into different lengths and labeled as follows: inoculation zone (i-zone), 0.5 m 2 , 1 m 2 , 1.5 m 2 and 2 m 2 ( Fig. 1). For the i-zone and for every 0.5 m 2 area, a 10 cm x 10 cm (100 cm 2 ) area was marked in the center of the defined lengths to recover spores from the surface. The entire Formica surface was sterilized by a three-step process. Progressively, the surface was cleaned with 7.0% hydrogen peroxide, 10% bleach and 70% ethanol. Following each of the first two disinfection processes, three rinses each with 250 ml of sterile distilled water was used to rinse the surface. This was followed by a final application of 70% ethanol. The Formica sheet was left to air-dry on a clean laboratory bench.
The C. difficile spore inoculum was prepared following EPA MLB SOP-MB-31 [EPA, 2017] and used to test the risk of cross-contamination by disinfectant wipes from the "i-zone" to Statistical analysis C. difficile spores were recovered from five test zones of a two-meter square Formica sheet and from used disinfectant wipes; counts were log 10 -transformed. Average log 10 CFU were calculated for wipes and defined sampled surfaces to test for statistically significant differences among eight disinfectant products. Specifically, we tested for differences among sampled surfaces by analyzing log 10 CFU/100 cm 2 counts recovered after disinfection. We also analyzed log 10 CFU/wipe used to test for the risk of crosscontamination from the i-zone to low risk surfaces. The least squares method of the Proc Glimmix test was used to fit liner models (n = 42, α = 0.05) and to test for interactions between disinfectant log 10 CFU/100 cm 2 and the surface area sampled. Surface area wiped and product type were treated as variables with continuous effects (repeated measures in Proc Glimmix). Tukey adjustments were used to test for significant differences in mean log 10 CFU among disinfectant products. The same procedure was also used to test for significant differences among surfaces treated with the same disinfectant wipe. All statistical tests were conducted using SAS version 9.4 (SAS institute, Cary, NC). Overall, and regardless of the sampling zone, the surface area wiped was statistically significant (P = 0.0001). On average, the log 10 CFU/100 cm 2 transferred to 0.5 m 2 and 1 m 2 surfaces from the i-zone were significantly lower compared to the log 10 CFU/100 cm 2 recovered from the i-zone post disinfection (P < 0.05). However, there were no statistically significant differences among the C. difficile spore log 10 CFU/100 cm 2 transferred to the 1.5 m 2 and 2.0 m 2 surfaces and the log 10 CFU/100 cm 2 detected from the i-zone after the wiping process (P ≥ 0.05). There were also no statistically significant differences among the mean log 10 CFU/100 cm 2 transferred to the 0.5 m 2 , 1 m 2 , 1.5 m 2 , and 2 m 2 surfaces from the i-zone (P ≥ 0.05).

T h e s p o r i c i d a l e f f i c a c y o f d i s i n f e c t a n t t o w e l e t t e s v a r i e s b y p r o d u c t t y p e a n d s u r f a c e a r e a
Overall, the product type was statistically significant (P = 0.0053). Specifically, SH was significantly more sporicidal than QA2 and QA3 (P < 0.05) as on average, lower log 10 CFU/100 cm 2 were recovered from the i-zone and subsequent surfaces (0.5 m 2 , 1 m 2 , 1.5 m 2 , and 2 m 2 ) (Fig. 3). However, there were no statistically significant differences among the sporicidal efficacies of SH, HP1, HP2, HP3 (Fig. 2) and QA1 (P ≥ 0.05; Fig. 3) as the mean log 10 CFU/100 cm 2 across the tested surfaces were very similar.
Surface area wiped was statistically significant (P < 0.0001) and overall, the sporicidal

Discussion
In this study, we determined the cross-contamination risk that disinfectant wipes may pose during and after the wiping process. We established that during the wiping process, disinfectant wipes transfer C. difficile spores from a contaminated surface (i-zone) to otherwise uncontaminated during the disinfection process. We also found that among all the used disinfectant wipes tested in this study, viable C. difficile spores were detected on the wipes post disinfection. Overall, we found that after the wiping process, the log 10 CFU/100 cm 2 detected from the 0.5 m 2 and 1 m 2 surfaces were significantly lower compared to those recovered from the i-zone. However, there were no significant differences among the log 10 CFU/ 100 cm 2 transferred to the 1.5 m 2 and 2.0 m 2 surfaces and the log 10 CFU/100 cm 2 recovered from the i-zone post-disinfection.

Disinfectant Wipes Cross-contaminate Hard Non-porous Surfaces
Cross-contamination is described by the CDC as the transfer of bacteria by contact from one surface to another [28]. Disinfectant wipes were the transfer "agents" between the surface inoculated with C. difficile spores and non-contaminated surfaces. In a similar study, Lopez et al. 2014 found that Bacillus thuringiensis spores inoculated on inanimate surfaces were transferred from wipe-disinfected fomites to fingers [29]. More recently, Becker et al. demonstrated that disinfectant wipes loaded with propanol or quaternary ammonium compounds (QAC) transferred viruses from a 25 cm 2 inoculated surface onto three other surfaces of the same size in the process of using the wipes [26].
Compared to the i-zone, the log 10 CFU/100 cm 2 from the 0.5 m 2 and 1 m 2 low risk surfaces were significantly lower than the log 10 CFU/100 cm 2 of the i-zone post disinfection. This could be explained by the observation that more disinfectant liquid was released from the wipe onto the 0.5 m 2 and 1 m 2 areas compared to the 1.5 m 2 and 2 m 2 areas. This was evident as the 0.5 m 2 and 1 m 2 surfaces were visibly wet after the wiping process. In a previous study by our group [22], we found that the percent of liquid released per 0.1 m 2 of a Formica surface significantly decreased as the surface area wiped increased.
However, overall, there were no significant differences in the log 10  The wipe design and substrate may also play a significant role in the level of organisms removed by the wipe [37]. Although the specific effects of the wipe materials were not evaluated in this study, differences in the levels of spores retained on the wipe could be associated with the wipe material type and with the amount of disinfectant liquid loaded on the wipe. Some wipe material types may hold more disinfectant liquid, which may be helpful in disinfection. We observed that wipes that had a rough feel probably due to their low cotton content (mostly the QA wipes) retained higher spore loads. In a 2012 study, Masuku et al. reported that the kind of material the wipe was made of, significantly impacted disinfection levels [38]. All hydrogen peroxide-based disinfectants tested in this study were more sporicidal than most (2/3) of the QAC disinfectant wipes tested. The sporicidal activity of hydrogen peroxides has been associated with their ability to produce free hydroxyl radicals after binding to deoxyribonucleic acids (DNA) [39]. These hydroxyl free radicals damage DNA and cell membrane lipids [39]. Although the tested disinfectants, with the exception of SH, had no sporicidal claims, we found that all the tested disinfectants reduced spore loads. This is likely a joint effect of physical spore removal by the wipe substrate and spore inactivation by the disinfectants [34,35,36]. Specifically, Rutala et al. reported that disinfectant wipes with no sporicidal claims had sporicidal effects, and the wipes could physically remove more than 2.9 logs of C. difficile spores from inoculated surfaces [35].
The US EPA requirements for obtaining a disinfectant label claim for C. difficile require a minimum of a six log 10 reduction [40]. But in our study, we found no statistical difference in disinfectant performance between all of the hydrogen peroxide wipes without a C.
difficile sporicidal label claim and the sodium hypochlorite-based product with a C. difficile sporicidal label claim. This suggest that the benefits in efficacy in passing the EPA method may not translate to actual differences in efficacy in real world use, as simulated in this study. Thus, there may be no actual clinical benefit from using a sporicidal disinfectant wipe in reducing patient risk of C. difficile infection versus using a hydrogen peroxide (non-sporicidal) disinfectant wipe. This needs further study.
We acknowledge that our study is limited as we did not investigate the effect the different wipe materials could have on the risk of cross-contamination. We also did not study the impact of a prolonged contact time on the inactivation of spores retained by used disinfectant wipes; both warrant further study.

Conclusion
Overall, disinfectant wipes may transfer C. difficile spores from contaminated to uncontaminated surfaces and retain high spore loads after the disinfection process, but the rate at which this occurs varies by product and likely is affected by the disinfectant liquid load, chemistry, and wiping material. We determined that non-sporicidal wipes reduce spore load, but the need to conduct similar studies using prevalent HAI pathogens Availability of data and material: All quantitative data generated or analysed during this study are included in this published article.
Competing interests: HFO, CAN, GKC, report grants from Diversey, Inc. during the conduct of the study. PT and XL report grants from Diversey, Inc. during the conduct of the study; personal fees from Diversey, Inc., outside the submitted work.
Funding: This work was supported by Diversey Inc., Charlotte, NC, USA.
Authors' contributions: CAN and GKC performed trials to develop the model, analysed and interpreted the data generated, and wrote the manuscript. XL provided industry experience, designed elements of the experimental protocol, and was a contributor in writing and editing the manuscript. PT also provided industry experience and was a contributor in writing and editing the manuscript. HFO served as the principle investigator for the study and was a contributor in writing and editing the manuscript. All authors read and approved the final manuscript.  Mean log10 CFU remaining on used wipes post disinfection with SH, hydrogen peroxide or quaternary ammonium alcohol disinfectant wipes