As the coronavirus disease 19 (COVID-19) pandemic appears here to stay, a new study published on the preprint server bioRxiv* in November 2020 reports an effective and rapid inactivation tactic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); namely, through the use of ozone water.
Preventing Viral Spread
The challenge of limiting transmission of the virus has fueled intensive research into different methods of viral inactivation. At present, most spread is considered to be via respiratory droplets emitted by infected individuals during sneezing, coughing, talking, singing, or even breathing. However, another important route of infection is by fomites – that is, contaminated surfaces or objects that have come into contact with the virus via incident infected droplets.
To prevent fomite transmission, hand cleansing using soap and water, or hand sanitizers containing 60% alcohol, are both recommended by the United States (US) Centers for Disease Control and Prevention (CDC). To be effective, hand washing should last 40-60 seconds, at least, based on the guidelines of the World Health Organization (WHO).
Many disinfectants have also been proposed to achieve rapid inactivation of the virus after a contact time of 30 seconds or more. One which has recently attracted attention is ozone water, already established as being capable of inactivating some microbes while being innocuous to humans. Nowadays, ozone generators that operate via electrolysis can produce water containing high ozone levels.
Measuring Ozone Water Inactivation
The current study is aimed at measuring the effect of ozone water on SARS-CoV-2 when used at various concentrations and for various contact times.
The viral strain used came from a patient on the cruise ship Diamond Princess, in February 2020, while the ozone water was used at concentrations of 1, 4, 7, or 10 mg/L. The contact time between the virus-containing solution and the ozone water was terminated at 5,10, or 20 seconds by adding sodium thiosulfate in an appropriate quantity.
Once this was done, serial dilutions of the virus solutions were inoculated on Vero cells and incubated for 72 hours, after 2 hours of adsorption. The cultures were then observed for cytopathic effects (CPE) in terms of the number of plaque-forming units (PFUs). Control virus suspension mixed with sterile tap water was also used.
Plaque formation in Vero cells. Virus solutions were exposed to ozone water for 5 sec and subjected to a plaque assay. (a) virus + tap water (no ozone), (b) virus + 7 mg/L ozone water or (c) mock-infected cells.
Dose-Dependent Inactivation of SARS-CoV-2
The researchers observed a marked CPE in the cells inoculated with the control suspension, while there was no discernible effect in the cells incubated with the ozone water-treated viral cultures at n SARS-CoV-2-infected cells, there was a marked cytopathic effect in cells inoculated with viruses without ozone water treatment (ozone water concentration of 7 mg/L ozone).
Thus, the sterile tap water did not inactivate the virus, but ozone water did in a dose-dependent fashion. The results were compared, following 5 and 10 seconds of reaction at each of the above concentrations.
The results showed that at concentrations of 1, 4, 7, and 10 mg/L, the ozone water produced a reduction in virus titer by ~81%, 93%, ~97%, and 97%, respectively, after a contact time of 5 seconds. At 10 seconds contact time, the reduction was almost identical.
The researchers observed that the concentration of ozone produced variations in the reduction in the viral titer. However, beyond 7 mg/L, increasing the concentration of ozone did not produce any significant effect on viral titer. The same is true of a contact time beyond 10 seconds. Thus, they say, “Taken together, ozone water showed a significant antiviral effect on SARS-CoV-2 in a dose-dependent manner. “
The study concluded that using ozone water containing high concentrations of ozone, at 7 or 10 mg/L, produced effective inactivation of the virus even when inhibitory matter like protein was present in the solution. Even at medium concentrations, such as 4 mg/L, ozone water is only a little less effective than alcohol-based hand sanitizers at hand disinfection. On the other hand, it is gentler on the skin.
Thus, the investigators comment, “The fact that higher concentrations of ozone water show stronger inactivating effects on SARS-CoV-2 may be a useful finding when using ozone-based hand disinfectants to prevent fomite infection.”
The question arises as to why a longer contact period failed to increase the inactivation effect. This is probably due to the completion of this effect within 5-10 seconds. However, it is also possible that the assay used is incapable of detecting reduction beyond this limit. A third possibility is that the culture medium diluted the ozone water. This was confirmed by the rapid decrease in ozone concentration to half in about 10 seconds. The effect was especially observed at lower concentrations of ozone.
The results of this study may be helpful as the world adjusts to the ‘new normal’ of life with COVID-19. An indispensable part of this is washing hands frequently and effectively. This is even more indispensable in healthcare settings, where the environment is often heavily contaminated with the virus.
The possibility of using high-concentration ozone water as a means of inactivating the virus on the hands, and thus preventing fomite transmission, is an important addition to the existing armamentarium of skin-compatible disinfectants - such as ethyl alcohol, para-chloro-meta-xylenol, and quaternary ammonium compounds. Not only does this leave the skin undamaged, making it suitable for sensitive skin, but it is sustainable and non-polluting even while it rapidly clears the virus.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.