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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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ACS Paragon Plus Environment


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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

274

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

275

recommendations respectively for the two cases where statistically significant differences were

276

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

277

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.

299 300

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

421 422



423

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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

424 425


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GRAPHICS

427

TOC Art.

428 429 430 431

432 433

Figure 1. Testing matrix.



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434 435

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.

445

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

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