Surface Cleaning and Disinfection: Efficacy Assessment of Four

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Surface cleaning and disinfection: efficacy assessment of four chlorine types using E. coli and the Ebola surrogate Phi6 Karin Gallandat, Marlene K. Wolfe, and Daniele S. Lantagne Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b06014 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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Environmental Science & Technology

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Surface cleaning and disinfection:

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Efficacy assessment of four chlorine types

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using E. coli and the Ebola surrogate Phi6

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Karin Gallandat1*, Marlene Wolfe1, and Daniele Lantagne1

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1: Department of Civil and Environmental Engineering, Tufts University, Medford, MA, USA

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*Corresponding author: 200 College Ave, Medford, MA 02155, USA.

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E-mail: [email protected]. Phone: +1 617 483 2045.

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ABSTRACT

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In the 2014 West African Ebola outbreak, international organizations provided conflicting

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recommendations for disinfecting surfaces contaminated by uncontrolled patient spills. We

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compared the efficacy of four chlorine solutions (sodium hypochlorite, sodium

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dichloroisocyanurate, high-test hypochlorite, and generated hypochlorite) for disinfection of

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three surface types (stainless steel, heavy duty tarp, and nitrile), with and without pre-cleaning

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practices (pre-wiping and/or covering) and soil load. The test organisms were E. coli and the

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Ebola surrogate Phi6. All tests achieved a minimum of 5.9- and 3.1-log removal in E. coli and

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Phi6, respectively. A 15-minute exposure to 0.5% chlorine was sufficient to ensure 0.05), except on heavy-duty tarp with soil load (p = 0.03).

appeared

equally

efficacious,

there

was

no

significant

difference

between

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A post-hoc analysis was conducted using Dunn’s test to compare chlorine types and

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recommendations respectively for the two cases where statistically significant differences were

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detected but no chlorine type or recommendation appeared to systematically perform better than

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the others (results not shown).

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Only NaDCC was used to evaluate the effect of a 15-minute exposure time and of a towel

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soaked in chlorine solution, as significant differences between chlorine type were not routinely

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identified. On heavy-duty tarp, increasing exposure time from 10 to 15 minutes did not

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significantly increase efficacy in any of the four recommendations with and without soil load, the

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only exception being in presence of soil load when doing nothing before applying chlorine

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(WMW test, p = 0.03). Applying a towel soaked in 0.5% NaDCC solution on the spill was not

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significantly more efficacious than using a dry towel to cover the spill on heavy-duty tarp

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(WMW test, p >0.05 both with and without soil load).

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Phi6 results.

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The surface carriers were inoculated with 8·107 CFU on average (range 1.3·106 to 2.3·108

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CFU). The recovery rates, as estimated based on the positive controls after 1-hour drying, were

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on the order of 20% on all three surfaces.

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All recommendations achieved at least 3-log Phi6 removal (Table 3). Out of a total of 288

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samples, Phi6 was detected after disinfection in six samples on nitrile and in no samples on

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stainless steel or heavy-duty tarp. The residual contamination in the positive samples ranged

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from 16 PFU/cm2 to 80 PFU/cm2 after disinfection. Due to the limited number of positive

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samples, statistical tests were therefore not conducted on Phi6 results.

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The geometric mean of triplicates exceeded the target of 10 residual PFU/cm2 in two instances

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(Figure 4): Phi6 was detected in presence of soil load when pre-cleaning nitrile discs with both

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generated and stabilized NaOCl.

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For the two conditions where Phi6 was detected on nitrile after a 10-minute exposure to 0.5% chlorine, increasing the exposure time to 15 minutes led to all non-detectable levels.

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DISCUSSION

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We conducted a systematic evaluation of the efficacy of surface disinfection recommendations

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for Ebola contexts, including testing two organisms, two soil load conditions, four surface

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cleaning recommendations, and four chlorine types on three surfaces commonly used in ETUs.

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Results indicated that test organism, surface type, and covering spills influenced surface

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disinfection efficacy, while chlorine type, soil load, and pre-cleaning did not. Overall, the

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recommendations provided during the 2014 EVD outbreak for surface disinfection appeared to

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be efficacious at removing E. coli and Phi6 from representative surfaces. Based on our results,

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we recommend a 15-minute exposure time to 0.5% chlorine to ensure safe surface disinfection in

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EVD contexts. These results: 1) are consistent with previous results while also providing further

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context-specific evidence; 2) answer questions about chlorine type, while raising questions about

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the role of soil load and how to appropriately model soil load in these contexts; 3) highlight the

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important role of test organism and surface type in disinfection; and, 4) inform how to conduct

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research on surface disinfection efficacy in further outbreak situations.

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The Cook et al.23,25 studies documented a 5-minute contact time with 0.5% sodium

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hypochlorite to completely disinfect stainless steel surfaces inoculated with the Ebola virus. Our

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data expand on their work as we found that 10 minutes were sufficient to disinfect stainless steel

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using Phi6 as an Ebola surrogate, but additional contact time was needed specifically on nitrile to

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ensure full disinfection under all tested conditions. This is also consistent with a previous study

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where environmental sampling was conducted in an isolation ward in Uganda in 2000. Bausch et

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al.13 found that CDC and WHO recommendations were efficacious, as Ebola was not isolated

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from surfaces after routine disinfection. However, the recommendations were more conservative

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during that time, as they recommended a 1% chlorine solution with a 15-minute contact time,

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and that a 10% chlorine solution should be used for heavy or dense spills.37 A more recent study

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conducted in Sierra Leone in 2015 suggests that environmental decontamination using 0.5%

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sodium hypochlorite was mostly effective at removing Ebola RNA from surfaces around the

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bedside, where contamination was detected most often.11 Our results also indicate 0.5% chlorine

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solutions should be efficacious.

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In contrast to beliefs that some chlorine types may be more efficacious than others, we found

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all chlorine types appeared equally efficacious for the disinfection of surfaces against E. coli and

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Phi6. We attribute this to the high chlorine dose (0.5%) applied. As such, we recommend using

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whichever chlorine source is available, safe to handle, and maintains an appropriate

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concentration.38

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The presence of soil load did not affect disinfection efficacy at a chlorine concentration of

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0.5%. We used the soil load mixture recommended in the ASTM International Standard26 and the

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chlorine demand of the spill with soil load represented less than 0.01% of the amount of applied

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chlorine. Again, we hypothesize soil load did not impact efficacy because of the high chlorine

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concentration applied. While this is likely representative of field practices, where chlorine will

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be generously applied to ensure disinfection, further research is needed to develop new matrices

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more representative of liquid Ebola bodily wastes and their impact on disinfection efficacy.

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Our results suggested that test organism and surface type do play an important role in

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determining disinfection efficacy. We found that E. coli was more challenging to disinfect on

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heavy-duty tarp, and Phi6 was more challenging to disinfect on nitrile; suggesting that different

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mechanisms are responsible for disinfection of the two organisms. A literature search provided

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little information on which fundamental mechanisms might explain the observed difference. We

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hypothesize that physical properties of the surface (such as roughness) as well as surface charge

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(of both the surface and the test organism) impacted disinfection efficacy. The fact that

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increasing the exposure time from 10 to 15 minutes did not improve disinfection efficacy against

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E. coli on heavy duty tarp suggests that bacteria did not enter in contact with the disinfectant,

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possibly due to some physical protection provided by the roughness of heavy duty tarp. It is

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unclear why such an effect would not be observed with Phi6, and additional research is indicated

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to investigate our hypotheses. The widespread use of both heavy-duty tarp and nitrile in

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emergency settings makes these results of particular concern. It is recommended to conduct

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future research with other surfaces commonly found in ETU settings, including paper, cardboard,

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cotton, rubber, latex, and concrete.

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The international recommendations for surface disinfection in Ebola contexts are particularly

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different in terms of practices. It has been suggested to wipe and to cover spills, and recently, to

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pre-clean surfaces with water and detergent.39 While pre-cleaning reduces the volume of spill to

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be disinfected, such a practice generates contaminated waste and can result in exposure of

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healthcare personnel to infectious materials. Although we were not able to test for the

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multiplicity of pre-cleaning practices that are likely to be encountered in the field (with or

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without soap, with dry and wet towels, and accounting for individual variability), our results

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suggest that wiping the surface before applying chlorine is unlikely to increase disinfection

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efficacy. This is consistent with a study of surface disinfection involving several viruses, where

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Tuladhar et al,.12 found that inactivation by a 0.1% chlorine solution was proportionally more

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important than the action of wiping. Covering spills is recommended to avoid disease

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transmission by splashes18 and our results with Phi6 suggest that it does not affect disinfection

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efficacy. We recommend covering spills with a cloth soaked in 0.5% chlorine and leaving it for

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15 minutes if disease transmission via splashes is a concern, as is the case with EVD due to the

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very low infectious dose.

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Our study had limitations, including that: 1) the comparison of E. coli and Phi6 is limited

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by different starting concentrations and detection limits; 2) although the recovery rates were

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similar for both organisms, we cannot rule out that some of the observed differences could be

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due to variability in recovery; 3) we used a surrogate organism instead of the actual virus, albeit

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one selected based on a comparison between the surrogate (tested in our laboratory) and Ebola

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virus (tested by Cook et al.23 in a Biosafety Level 4 laboratory); 4) we believe that our protocol,

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using 8-cm in diameter discs instead of the 1-cm diameter surface carriers recommended in the

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ASTM Standard,33 simulates field conditions more accurately, however 2-mL spills are still

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unlikely to reflect the extent of environmental contamination experienced in ETUs; 5) our

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standardization of pre-cleaning recommendations does not account for further variability in pre-

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cleaning practices applied in ETU settings; 6) the ASTM soil load might not be representative of

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the matrices in which the Ebola virus would be shed by patients; and, 7) temperature and relative

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humidity in the laboratory were both lower and more controlled than those commonly found in

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ETU settings.

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Despite these limitations, we feel our data contribute to the understanding of, and assist in

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providing an evidence base for, surface disinfection practices in outbreaks. In particular, our

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results support the recommendation of a 15-minute exposure to 0.5% chlorine – independently of

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chlorine type, surface type, practices, and presence of organic matter – as an efficacious measure

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to interrupt EVD transmission via fomites. Using Phi6 as a surrogate allowed us to carry out

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extensive testing and to identify critical conditions; we recommend evaluating the resistance of

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the Ebola virus (at Biosafety Level 4) to a 15-minute exposure to 0.5% chlorine, without pre-

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cleaning or covering, with or without soil load, on nitrile and, if possible, on other surfaces, to

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confirm the results presented here.

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Further research is required to investigate how surface properties and complex matrices affect

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disinfection efficacy, as well as to understand the role of wiping or covering spills. A more

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fundamental understanding of the mechanisms affecting disinfection efficacy and of the physico-

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chemical interactions between microorganisms and surfaces will allow extension of these results

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to different pathogens with more flexibility.

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Our study was carried out in response to the 2014 Ebola outbreak and focused on evaluating

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the efficacy of existing recommendations in place during that outbreak. It is recommended future

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research be completed to develop appropriate recommendations, not just test the efficacy of

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existing recommendations. For instance, using NaDCC granules instead of a liquid chlorine

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solution to cover uncontrolled spills has been identified as an efficacious procedure against

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bacteria in developed country hospitals.29 Additional research is needed to evaluate the adequacy

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of this, and other novel, protocols as a possible intervention in ETU contexts.

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For diseases with infectious doses as low as EVD, multi-barrier approaches (including

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personal protective equipment, handwashing, disinfection of surfaces and safe waste

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management) should be used to minimize transmission risk. However, multi-barrier approaches

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– particularly those with waiting times – are burdensome in the ETU context, due to the

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maximum time responders can spend in PPE. A broader reflection around the best way to

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minimize transmission risk and the appropriate number and level of barriers to use in healthcare

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facilities and communities facing situations such as EVD is necessary. For example, it has been

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suggested to depend on PPE primarily, and not be concerned with disinfecting spills in the ETU

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context. Ideally, further discussion will involve responders, members of the scientific community

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and international organizations to address the appropriateness of existing recommendations and

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how they can be adjusted to increase preparedness for future outbreaks.

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TABLES

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Table 1: Recommendations on surfaces disinfection in Ebola outbreaks.

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Target

Action

Disinfectant

Exposure time

Source

Hospital/ETU

Pre-clean surface

0.5% chlorine

10 minutes

WHO19

Household

Cover spills

0.5% chlorine

15 minutes

CDC40

Hospital

Pre-clean surface

“Chemical disinfectant for non-enveloped viruses”

Not specified

CDC20

ETU

Do nothing

0.5% chlorine

15 minutes

MSF18

Table 2: Chlorine types commonly used in emergency contexts.

Chlorine type

Expected pH

Form

Advantages

Drawbacks

Sodium dichloroisocyanurate (NaDCC)

6

Granules

Easy to ship Long shelf-life Does not clog pipes

Smell

High-test hypochlorite (HTH)

11

Granules

Easy to ship Long shelf-life Does not clog pipes

Explosive Clogs pipes

Stabilized sodium hypochlorite

11

Liquid

Can be local Does not clog pipes

Shorter shelf-life Difficult to ship

9-11

Liquid

Can be on-site Does not clog pipes

Shorter shelf-life Difficult to ship Quality control?

Non-stabilized sodium hypochlorite

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Table 3. Observed log removals and standard deviations.

Surface type

Soil load

Rec.

E. coli log removal

STD (E. coli)

Phi6 log removal

STD (Phi6)

Do nothing

>6.6

0.0

4.1

0.0

Wipe

>6.6

0.0

4.1

0.0

Cover

6.0

0.4

3.4

0.0

Wipe & cover

6.0

0.3

3.4

0.0

Do nothing

>6.8

0.0

5.7

0.0

Wipe

>6.8

0.0

5.7

0.0

Cover

>7.3

0.0

5.5

0.0

Wipe & cover

7.2

0.2

5.5

0.0

Do nothing

6.4

0.0

4.8

0.0

Wipe

>6.4

0.0

4.8

0.0

Cover

6.4

0.2

3.1

0.0

Wipe & cover

>6.5

0.0

3.1

0.0

Do nothing

>6.9

0.0

3.6

0.0

Wipe

6.8

0.2

3.3

0.4

Cover

>6.8

0.0

5.5

0.0

Wipe & cover

6.7

0.4

5.5

0.0

Do nothing

6.7

0.8

3.9

0.1

Wipe

6.7

0.9

3.8

0.0

Cover

6.3

0.9

3.4

0.0

Wipe & cover

6.1

1.0

3.4

0.0

Do nothing

6.0

1.1

5.4

0.0

Wipe

5.9

1.0

3.2

0.0

Cover

6.4

0.9

5.5

0.0

Wipe & cover

6.3

0.9

5.5

0.0

Without

Stainless steel

With

Without

Nitrile

With

Without

Heavy duty tarp

With

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GRAPHICS

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TOC Art.

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Figure 1. Testing matrix.

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Figure 2: Number of CFU E. coli per unit area detected after disinfection.

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The points correspond to the geometric mean of triplicate samples. Error bars represent standard errors of the mean. “ND” stands for “not detected”, and the theoretical detection limit was < 1 CFU/cm2. The points indicated as too numerous to count (TNTC) correspond to cases were all triplicates were TNTC after disinfection.

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Figure 3. Statistical results of E. coli testing.

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NSD stands for “no significant difference” based on the Kruskal-Wallis test (p