key: cord-0945039-oub2pdms authors: Huang, Yi; Xiao, Shuqi; Song, Donglin; Yuan, Zhiming title: Evaluating the Virucidal Activity of Four Disinfectants Against SARS-CoV-2 date: 2021-11-12 journal: Am J Infect Control DOI: 10.1016/j.ajic.2021.10.035 sha: 4a03e6cb31a8ff8d833a81a471a45641bb02f919 doc_id: 945039 cord_uid: oub2pdms Background:The recent COVID-19 pandemic highlights the need for efficacious virucidal products to limit the spread of SARS-CoV-2. Several studies have suggested that alcohol-based sanitizers and some disinfectants are effective. While virucidal activity data of low-level disinfectants are lacking and some conclusions are not clear yet. Methods:We evaluated the virucidal activity of two quaternary ammonium compounds (QAC) disinfectants (MICRO-CHEM PLUS and FWD), W30 (an amphoteric surfactant) and Medical EtOH® against SARS-CoV-2. Suspension tests covering different concentration and contact time were performed using the integrated cell culture-qPCR method. Results Each of disinfectants was effective at inactivating SARS-CoV-2. MCP and FWD are highly effective within 15 seconds. W30 is also efficient within 2 minutes at concentration of 1%. Consistent with previous report, our results also demonstrated that 38% ethanol was sufficient to completely inactivate virus, which proved the method used in this study is feasible. Conclusion and Discussion QAC disinfectants, MCP and FWD, are highly effective for the inactivation of SARS-CoV-2, which making them practical for use in healthcare setting and laboratories where prompt disinfection is important. The low-level disinfectant based on amphoteric surfactant, W30, which may present in commonly available household hygiene agents is also able to inactivate SARS-CoV-2. An emergent pneumonia outbreak happened in the late December 2019. Researchers have quickly isolated a new virus from the patient and sequenced its genome 1 . The infectious agent of this disease was identified as a novel coronavirus, SARS-CoV-2, and the novel viral pneumonia was named as COVID-19 by WHO. The current COVID-19 pandemic in more than 250 countries has become a serious threat to the public health and economy worldwide. The novel coronavirus belong to the family of Coronaviridae (β-CoV) together with SARS-CoV and MERS-CoV according to the phylogenetic analysis based on the viral genome 1, 2 , which comprising large, single, plus-stranded RNA genome, but the nucleotide sequence similarity is less than 80% between SARS-CoV-2 and SARS-CoV (about 79%) or MERS-CoV (about 50%). Unlike SARS-CoV, SARS-CoV-2 seems to replicate efficiently in the upper airways during the incubation period, which is estimated to last up to 14 days 3, 4 . During the prodromal stage, 4 asymptomatic and pre-symptomatic individuals release large amounts of viruses from infected cells 4 . As a result, viral transmission is more effective with SARS-CoV-2 than with SARS-CoV. The rapidly increasing number of cases and evidence of human-to-human transmission also suggested that SARS-CoV-2 was more contagious than SARS-CoV and MERS-CoV 5, 6 . SARS-CoV-2 is mainly transmitted through person-to-person close contact (<1.5-2.0 m), as well as by aerosol respiratory droplets smaller than 5 μm of diameter 7 . The transport of droplet aerosols generated by infected individuals is an issue of considerable concern and importance and has been taken into account to reduce the risk of infections 8, 9 . The common transmission routes include direct transmission (cough, sneeze, and droplet inhalation transmission) and indirect transmission. Among many sources of indirect transmission, there is the contamination of inert/inanimate surfaces and hands. SARS-CoV-2 may be transmitted via contact by touching contaminated surface, followed by touching mouth, nose or eyes. Experimental studies have reported prolonged survival of SARS-CoV-2 on inanimate surfaces and objects under laboratory conditions (e.g., a large inoculum of 10 7 virus particles on a small surface), and the conclusion was that fomite transmission of SARS-CoV-2 is certainly plausible 10 COVID-19 has been particularly devastating, thus enhanced disinfection and other preventative measures against SARS-CoV-2 have been adopted worldwide to limit its spread. For example, WHO recommends cleaning surfaces with water, detergents and disinfectants usually effective to clean the environment 12 , because SARS-CoV-2 should be very susceptible to most cleaning agents as an enveloped virus. Some studies using other coronaviruses as the surrogate performed in the last decades have reported the effects of a number of disinfectants for the mitigation of the coronavirus: the ethanol at concentration >62%, isopropanol, povidone iodine, sodium hypochlorite and quaternary ammonium compounds combined with alcohol are effective for surface disinfection [13] [14] [15] [16] . In turn, hydrogen peroxide vapor, chlorine dioxide, ozone, and UV light could be applied to reduce viral load present in aerosols [17] [18] [19] [20] . And several recent studies have performed the in vitro evaluation of disinfection effectiveness against SARS-CoV-2 suggested that alcohol-based disinfectants such as ethanol and isopropanol, and some alcohol-free hand sanitizer and UV light are really effective against SARS-CoV-2 16, 21-23 . Nevertheless, the disinfection data for SARS-CoV-2 are still limited as a novel virus and a biosafety level-3 (BSL-3) agent. We could draw conclusions about which disinfectants are effective against it from the studies using other coronaviruses speculatively, while even viruses within the same family can respond differently to a 6 given disinfectant 24 . In addition, there is some divergence about whether some disinfectants work best against SARS-CoV-2. For instance, one prominent review article reported that benzalkonium chloride was probably "less effective" against SARS-CoV-2, which was cited by the Centers for Disease Control (CDC) of the United States as a reason to avoid using benzalkonium chloride-based hand sanitizer products [25] [26] . At the same time, the Environmental Protection Agency (EPA) of the United States and Health Canada both list benzalkonium chloride product on their official list of disinfectants recommended for use against SARS-CoV-2 27 . More research is needed in this area. In this study, we tested two quaternary ammonium compounds (QAC) Virucidal products tested 7 Four disinfectants were tested in this study (Table I) . Among them, Micro-Chem Plus® (MCP, National Chemical Laboratories, Inc., Philadelphia, Pennsylvania) and Medical EtOH® are two commercial, broad-spectrum disinfectants. Similar with MCP but more environmental friendly, FWD is also a dual quaternary ammonium compounds product which is still in the stage of research and development. W30 is a raw material on the basis of an amphoteric surfactant for use in biocidal products which core ingredient is. N-Alkyl aminopropyl glycine. Table 1 . Tested disinfectants list and the cell sensitivity to the disinfectants. "+" means cytotoxicity; "-" means no cytotoxicity. * A slight cytotoxic effects caused by medical ethanol was observed, but it disappeared after dilution was discarded and cells were overlaid with fresh DMEM with 2% FBS medium for overnight. Most disinfectants destroy cell cultures and the mixtures of viruses and disinfectants must be diluted before testing. So cytotoxic effects were firstly assessed in Vero cells using medium (DMEM with 2% FBS) and disinfectant but without addition of virus to ensure that diluted disinfectants was not cytotoxic. Briefly, the test products were serially diluted, and aliquots of 1mL from each sample were inoculated into cells. Following for 1h incubation at 37°C, dilutions were discarded and cells were overlaid with fresh medium. The cells were observed for cytotoxic effects for the same 9 incubation time which was later used for the suspension tests (Table 1) . A suspension test method was used for inactivation assay in this study 28 were fixed with 4% paraformaldehyde for 1h to inactivate infectious virus. Cells were stained with 1% crystal violet, and plaques were visualized and counted. The virucidal activity was determined by the difference of the logarithmic titre of the virus control minus the logarithmic titre of the test virus, reduction factor (RF). The Log10 titre and its standard deviation (SD) were calculated as well as the variance of the RF. RF of ≥4 was regarded as evidence of sufficient virucidal activity 28 . Full results of suspension tests are listed in Table 2 . Each of disinfectants tested was effective at inactivating SARS-CoV-2. 2) . Based on the data obtained above we compared the inactivation profiles of four disinfectants ( Table 2) source not found. and demonstrated that this method is a feasible strategy. In addition, we also performed the plaque assay to calculate the residue virus and obtained the same results with the integrated cell culture-qPCR method (Fig. 3) . 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Beijing: Ministry of Health of the People's Republic of China The relationship of concentration and germicidal efficiency of ethyl alcohol Factors affecting stability and infectivity of SARS-CoV-2 Hand sanitizers: a review of ingredients, mechanisms of action, modes of delivery, and efficacy against coronaviruses EPA Approves 13 Products from List N as Effective Against SARSCoV-2. 2020. Available at Antisepsis, disinfection, and sterilization Increased use of quaternary ammonium compounds during the SARS-CoV-2 pandemic and beyond: consideration of environmental implications We thank Dr. Xiaoxiao Gao for her advices on experiment design and all colleagues from National Biosafety Laboratory (Wuhan), CAS for their support during the study. This work was supported by Advanced Customer Cultivation Project of Wuhan National Biosafety Laboratory, Chinese Academy of Sciences (2019ACCP-MS08). All authors report no conflicts of interest relevant to this article.