key: cord-311012-wyglrpqh
authors: Meyers, Craig; Kass, Rena; Goldenberg, David; Milici, Janice; Alam, Samina; Robison, Richard
title: Ethanol and Isopropanol Inactivation of Human Coronavirus on Hard Surfaces
date: 2020-09-28
journal: J Hosp Infect
DOI: 10.1016/j.jhin.2020.09.026
sha: 
doc_id: 311012
cord_uid: wyglrpqh

BACKGROUND: The COVID-19 pandemic has greatly increased the frequency of disinfecting surfaces in public places causing a strain on the ability to obtain disinfectant solutions. An alternative is to supply plain alcohols (EtOH and IPA) or sodium hypochlorite (SH). AIM: There are few data showing the efficacy of multiple concentrations of EtOH, IPA, and SH on a human coronavirus (HCoV) dried on surfaces using short contact times. METHODS: Multiple concentrations of EtOH, IPA, and SH to inactivate high numbers of HCoV under real-life conditions were tested. High concentrations of infectious HCoV were dried on porcelain and ceramic tiles, then treated with multiple concentrations of the alcohols for contact times of 15 sec, 30 sec, and 1 min. Center for Disease Control (CDC) recommended three concentrations of SH were also tested. Reductions in titres were measured by using the tissue culture infectious dose 50 (TCID(50)) assay. FINDINGS: Concentrations of EtOH and IPA from 62% to 80% were very efficient at inactivating high numbers of HCoV dried on tile surfaces even with a 15 sec contact time. Concentrations of 95% dehydrated the virus, allowing infectious virus to survive. The CDC recommended 1/10 and 1/50 dilutions of SH were efficient at inactivating high numbers of HCoV dried on tile surfaces, whereas, a 1/100 dilution had substantially lower activity. CONCLUSIONS: EtOH, IPA, and SH at multiple concentrations efficiently inactivated infectious virus on hard surfaces, typical of those found in public places. Often no remaining infectious HCoV could be detected.

With the rapid spread of SARS-CoV-2 around the world, there was a concomitant rapid increased need of effective sanitizers/disinfectants. With the major method of transmission being through aerosolized respiratory droplets, and virus on surfaces remaining viable for hours and even days 1-3 , there arose a constant need to properly disinfect surfaces. Healthcare institutes, businesses, and homes quickly found themselves in need of reagents to disinfect surfaces. During this pandemic, the necessity to disinfect surfaces at an increased rate resulted in product shortages. The Environmental Protection Agency (EPA), in order to make everyone aware of, and have access to, potentially effective surface disinfectant products for use against the possible presence of SARS-CoV-2 on surfaces, provided a list of "Disinfectants for Use Against SARS-CoV-2" 4 . Under their guidelines, the EPA allows a manufacturer to provide data that shows that their product(s) is (are) potentially effective against harder-to-inactivate viruses. After receiving approval from the EPA, the manufacturers can market claims for use against SARS-CoV-2. While this list can potentially provide important information to users it has several shortcomings. First, for many of the disinfectants on the list, it is difficult, if not impossible, to obtain the experimental protocols used to determine the effectiveness of the disinfectant. Second, the FDA, EPA, and the Department of Health and Human Services (DHHS) all require that standards for meeting efficacy data requirements include testing virucidal effects on a carrier (surface), as opposed to a suspension assay [5] [6] [7] . Third, the surface or carrier test also requires the drying of virus onto the carrier in the presence of a protein "soil"; then the virus is recovered and assayed for infectivity [5] [6] [7] . Fourth, many of the contact times reported on the EPA's list are not realistic for practical use. A contact time of over 1 min is often not pragmatic under normal situations in healthcare institutes, business, or homes. It is also questionable if the viruses used to make these claims are really harder-to-inactivate viruses. This list was developed to provide needed guidance at the beginning of a situation, such as the present pandemic. For that early need, it was an important aid. However, the list was meant to be a stop gap until more evidence-based studies could be completed.

With the rapid loss of ready available disinfectants, many public places have found that the only products available in needed quantities are common alcohols, ethanol (EtOH) and isopropyl alcohol (IPA), without additives. Here we demonstrate the efficacy of EtOH and IPA, two basic disinfectants that are relatively easy to obtain, and are relatively safe for use in public areas. We tested a wide variety of concentrations of the two alcohols commonly used in disinfectants and sanitizers. To satisfy the requirements of hard surfaces as carriers, we used ceramic and porcelain tiles that are commonly found in public places. All assays were performed in the presence of a 'soil', bovine serum albumin (BSA). For all of our assays, we used high titre stocks of HCoV-229e, as a surrogate for SARS-CoV-2. While there are clear differences in the pathology of these viruses, they are in the same virus family, having very similar structures, and both are human respiratory pathogens. SARS-1 has been shown to survive longer in a dried state for up to 9 days where HCoV-229e survived only up to 6 days 8 , but disinfectant comparisons were not performed. Using the surrogate has allowed us to rapidly test and provide science-based answers in the search for an effective surface disinfectant, using contact times that better reflect real-life situations.

Huh7 cells were grown in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (DMEM10) and 100 U/ml pen/strep, and the cells grown in 5% CO 2 at 37˚C. Infectious stocks of Human Coronavirus 229e (HCoV 229e) were prepared by seeding T75 flasks with 7 x 10 6 Huh7 cells, which were incubated overnight. On the following day, the media was changed to DMEM with 2% FBS (DMEM2) and a cells were infected with virus using a multiplicity of infection (MOI) of 0.01. The infected flasks were incubated for two days in 5% CO 2 at 35˚C. On the second day, the flasks were frozen at -80˚C for at least 1 h, then thawed in a 37˚C water bath taking care to remove them from the water bath before they were completely thawed. Thawing was then completed at room temperature. The cell suspensions were transferred to a 15 ml polypropylene tube and sonicated on ice in a cup sonicator at 100 watts peak envelope power, 3 bursts of 20 s each. The lysates were clarified by centrifugation at 3,000 rpm for 10 min at 4˚C, and the supernatant poured into a fresh 15 ml tube. Virus solutions were aliquoted into 8-0.5ml portions, and several smaller aliquots were then frozen for long term storage at -80˚C. One of the smaller aliquots was used to determine the titre of the stock by the tissue culture infectious dose 50 (TCID 50 ) assay.

Huh7 cells were harvested, counted, and resuspended into DMEM2 to a concentration of 1.5 x 10 6 cells/ml. Then, 100 µl of the cell suspension was added to each well of the 96-well plate. Plates were incubated overnight in 5% CO 2 at 37˚C. Serial 10-fold dilutions of virus were added to each column of wells containing cells. An extra row of mock-infected cells were included across the bottom as controls. The plates were then incubated for 3 days in 5% CO 2 at 35˚C. On the third day, the wells were examined for the presence of cytopathic effects (CPE) and the TCID 50 calculation was done using the Reed-Muench method 9 , based off the number of wells positive for CPE at each dilution.

The disinfectants used in the study were ethanol (EtOH) (Fisher Scientific), isopropanol (IPA) (Sigma -Aldrich), glutaraldehyde (GTA) (Cidex and Cidexplus; Advanced Sterilization Products), orthophthalaldehyde (OPA) (Cidex OPA; Advanced Sterilization Products), and sodium hypochlorite (SH) (Activate; Deardorff Fitzsimmons Corp.). Each disinfectant was prepared according to manufacturer's recommendation and stated dilution immediately before testing.

Porcelain and ceramic tiles were purchased from a local building supply company and used as carriers for the disinfection assays (Fig 1) . Carriers were prepared by soaking in 10% hydrogen peroxide for 15 min, neutralization in sterile water containing 200 U/ml of catalase for 10 min, and J o u r n a l P r e -p r o o f rinsing in sterile water for 10 min before being dried in a sterile petri dish 10, 11 . Two hundred µl of an organic load or soil of 5% BSA was added to the virus suspension and 200 µl of this virus solution was spread onto a single carrier side with a sterile pipette tip. The inoculated carriers were allowed to dry in a laminar flow cabinet for 30 min. Immediately after drying, 1 ml of the liquid disinfectants were added to the surface of the carrier, covering the entire area containing the dried virus. Carriers were then incubated at room temperature for contact times of 15 s, 30 s, and 1 min. Virus was then scraped off the carrier into a 15-ml Amicon Ultra centrifugal filter (100,000 MW cut-off [MWCO]) and immediately 2 ml of neutralizer was added to the filters. The filters were centrifuged at 4,000 rpm for 10 min. The filters were washed a total of 4x with DMEM2 and centrifuged at 4,000 rpm for 10 min. The virus-containing eluents were then assayed for infectivity using the TCID 50 method. At least four replicate assays were done for each disinfectant and contact time. Controls for virus recovery after drying on a carrier were included for every set of assays performed. In every case, we never observed a significant decrease (<0.5 log 10 ) in infectious virus due to drying the virus on either type of tile. Neutralizers used were DMEM2 for EtOH and IPA, and 7% glycine for SH, GTA, and OPA. Table I shows that in general, both EtOH and IPA were highly effective at inactivating HCoV. EtOH concentrations of 62%, 70%, 75%, and 80%, and IPA concentrations of 70%, 75%, and 80% were all able to produce greater than 4 log 10 inactivate of HCoV, and in some cases, we were unable to detect any remaining infectious virus. Greater than 99.99% reduction of infectious virus was observed at all contact times, including the shortest time of 15 s. In many instances, we were unable to detect any residual infectious virus (Table I) . However, in a few cases with contact times of 15 or 30 s, some of the replicates reached only a 3 log 10 reduction (Table I) , although this level still means that over 99.9% of the infectious virus was destroyed. Interestingly, at the highest concentrations tested, 95% EtOH and 95% IPA, we observed significant reductions in inactivating, with some contact times producing less than a 2 log 10 reduction of infectious virus.

Because of our previous experience in testing bleach (SH) [10] [11] [12] [13] , and the potential for people to have it available for use in disinfecting surfaces, we decided to include SH in these evaluations. Common concentrations of bleach purchased by the general public contain between 5% and 6% SH, and it is recommended to be used at a 1/10 to a 1/100 dilution. We used 5.25% SH diluted 1/10 (~0.525%) as we have done previously as a positive virucidal control 10, 11, 13 . As expected, 0.525% SH was highly effective at inactivating HCoV, producing greater than a 4 log10 decrease in infectious virus. We were unable to observe any sign of remaining infectivity at all contact times of 1 min, 30 s, and 15 s (Table  I) . Because there are recommendations to use bleach at 1/50 and 1/100 dilutions for sanitizing surfaces 14, 15 , we decided to also test the 1/100 dilution. At a 1/100 dilution or ~0.0525% SH, we observed significant decreases in its ability to inactivate HCoV (Table I) . Sometimes, the inactivating dropped below a 2 log 10 decrease. We also tested a 1/50 dilution or ~0.1% SH (Table I ). The efficacy of 0.1% SH was similar to that of 0.525% SH, both producing greater than a 4 log 10 decrease in infectivity with contact times of 30 s and 15 s. With both 0.525% and 0.1% SH there was no evidence of any remaining infectious virus (Table I) .

Because of its prominent role for the last several decades as a hospital sterilant, we also tested J o u r n a l P r e -p r o o f (GTA). As expected, GTA proved to be highly effective at inactivating HCoV, producing greater than 4 log 10 decrease in infectious virus at all contact times (Table I) .

In the midst of a pandemic, the supply chain may not be able to provide traditional disinfectants fast enough to meet needs. Basic alcohols, EtOH and IPA, are likely easy solutions to fill unmet needs. Clear evidence-based answers to important questions of contact times, effect of carrier types, and the influence of soil contamination on disinfectants used on a new infectious organism are minimal or nonexistent. Our goal was to create a more real-world situation to evaluate the efficacy of available disinfecting agents. As we are finding out in the present pandemic situation, these may be the only products available during times of shortages. We used a wide range of concentrations, porcelain and ceramic tile carriers, which are common surfaces in public places, and contamination with organic soil, and short contact times that represent the reality of practices used in disinfecting public spaces. Our studies demonstrate that EtOH and IPA at concentrations ranging from 62% to 80% are highly effective at inactivating HCoV on tile surfaces even with contact times as low as 15 sec. While we saw slight differences between the two carriers, these were inactivating over 99.99% or 99.9% of the virus. Noteworthy differences were observed when we used 95% EtOH or 95% IPA. It is likely that a minimum concentration of water is required to catalyze microbial penetration, allowing these agents to optimally perform their destructive effects. Higher concentrations may dehydrate the virus, allowing significant levels to remain viable 16, 17 . Therefore the adage that "more is better" is not necessarily true for alcohols. Because many public spaces have bleach on hand, we also tested bleach at three recommended concentrations 14, 15 . Concentrations of 0.525% and 0.1% both inactivated 99.99% or more of the virus within short contact times. However, 0.0525% SH was much less effective at inactivating the virus.

Several studies have reported the efficacy of various disinfectants in inactivating CoV, including animal 18-23 and human [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] CoV. These studies are discussed in a 2020 review 11 . The majority of these studies did not use the carrier method of drying the virus onto a solid surface. It has been recently shown that HCoV can remain infectious in aerosols for hours and on surfaces for days 12 , demonstrating the importance of testing the efficacy of a disinfectant in the manner in which it will be used, i.e., to disinfect solid surfaces. Of those studies that used the carrier method, some dried the virus on petri dishes 2,13 , while others used stainless steel as their carrier 1, 14, 15 . Similar to the use of stainless steel, we used carriers that represented real-life situations. Porcelain and ceramic tiles are common surfaces in many public places, and during a pandemic, would be surfaces that require constant disinfection. In our study, we saw only small effects, of less than 1 log 10 of inactivating, between the two tile types, for only a few of the disinfectant concentrations and contact times tested.

Previously published studies particularly relevant to our results used different concentrations of EtOH and IPA [7] [8] [9] 15, 16 . All but one of these studies used the suspension technique, so those testing high concentrations of EtOH or IPA were unable to factor in the rapid dehydration phenomenon which is characteristic of these agents. The only study that performed testing by drying the virus on a carrier, only used 70% EtOH 15 . Results from these studies aligned well with our results presented here, but none covered the range of concentrations we covered, with the majority only testing a few concentrations. In addition, only one study used the carrier model. Two studies that tested bleach as a surface disinfectant both showed, as we did, that diluting SH 100-fold resulted in poor inactivating of two animal CoV, mouse hepatitis virus and transmissible gastroenteritis virus 15 , or inactivating HCoV 229e 1 . In addition, one of these studies also tested SH diluted 50-fold and observed a 99.9% reduction in virus titre 1 similar to the results we observed.

SARS-CoV-2 is the third pathogenic HCoV with significant mortality in the past 20 years to spread in the human population 17, 18 . After its emergence, it quickly spread around the world. While there is a potential for other mechanisms of transmission, aerosolization and fomites are considered the most probable means. Some of the most common symptoms of SARS-CoV-2 disease are associated with the formation of aerosols. Persons infected but showing only mild or no symptoms can also readily spread the virus by aerosols 19 . Aerosols directly and indirectly readily contaminate surfaces in all public places, on which the virus can stay infectious for days 12 . This resulted in persons responsible for the safety of many public places, including healthcare institutes, businesses, and homes, trying to find solutions to their need to disinfectant surfaces much more often. This led to a rapid strain on the supply chain in providing sufficient quantities of normally used disinfectant compositions. One answer was the use of readily available agents such as common alcohols (EtOH and IPA) and diluted bleach. This study provides important information on the efficacy of these agents at different concentrations to inactivate HCoV. 

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None of the authors have any financial and personal relationships with other people or organizations that could inappropriately influence or bias of this work.

This study was funded in part by the Huck Institutes of the Life Sciences' Coronavirus Research Seed Fund (CRSF).