Sir,
SARS-CoV-2, the causative agent of COVID-19, is transmitted via the aerosol route and from larger sprayborne droplets from infected individuals [1]. Infectious particles generated by an individual will be, in the case of exhaled aerosols or nasopharyngeal secretions, associated with high salt and proteinaceous matrices which can provide protection to the agent against environmental stresses and decontamination methods [2]. Gaseous decontamination techniques are widely used in healthcare laboratories and more recently have been proposed as a method for decontamination of single-use face masks to avoid supply shortages [3,4]. The current study investigated the inactivation of SARS-CoV-2 dried from multiple media using a commercial vapourized hydrogen peroxide (VHP) generator (X10; Steris, Basingstoke, UK).
SARS-CoV-2, England 02/2020 (EPI_ISL_407073), was propagated as described by Paton et al. [5]. The viral stock was used to create two separate suspensions for exposure: (A) diluted 1:1 with complete minimum essential medium (CMEM); (B) diluted 1:1 with artificial saliva (BS EN 16711-3:2019) with additional protein (mucin (2.5 mg/mL) and bovine serum albumin (2.0 mg/mL)). Suspension A or B (10 μL) was dried on to stainless steel coupons (15 mm diameter) within an operating class III biological safety cabinet for ∼2.5 h. Coupons were then transferred to the flexible film isolator (FFI, 488 L) for exposure. The X10 generator was connected to the FFI and the smallest preset cycle started; triplicate zero-minute time-point coupons were sampled immediately taken by placing each into 1 mL of CMEM plus catalase (VWR, Lutterworth, UK). Samples were further taken at 5 and 10 min after VHP exposure. Inoculated coupons not exposed to VHP were also made to assess the loss in recovery over the exposure period. Samples were then removed from the FFI and transferred to a class III BSC for processing. Once in the BSC, the drying control coupons were placed into recovery media and all coupons were processed as described previously by serial dilution and plaque assay using Vero E6 cells [5].
After exposure to VHP, no viable SARS-CoV-2 (detection limit 2.5 pfu in each sample) was recovered from any exposure samples taken, producing a reduction in comparison to the loading at time zero of >3.6 log10 for SARS-CoV-2 exposed in saliva and protein and >4.1 log10 from SARS-CoV-2 exposed in CMEM (Table I). The concentration of hydrogen peroxide within the FFI was measured at a peak of 726 ppm at 150 s.
Table I.
Recoveries and log10 reductions for the sample points during the exposure cycle of vapour hydrogen peroxide
| Sample point (min) | Complete MEM |
Synthetic saliva + protein |
||
|---|---|---|---|---|
| Recovery/pfu (SE) | Log10 reduction | Recovery/pfu (SE) | Log10 reduction | |
| 0 | 2.8 × 104 (1.6 × 104) |
– | 9.7 × 103 (6.7 × 102) |
– |
| 5 | ND <2.5 | >4.1 | ND <2.5 | >3.6 |
| 10 | ND <2.5 | >4.1 | ND <2.5 | >3.6 |
MEM, minimum essential medium; pfu, plaque-forming units; SE, standard error; ND, none detected.
Hydrogen peroxide technologies have been evaluated previously for their ability to inactivate SARS-CoV-2 dried on to surfaces [6,7]. These studies used SARS-CoV-2 virus suspended in its propagation media, or in other soil, which does not replicate the presentation of the virus on used face masks from exhaled or sprayborne droplets [6,7]. To the authors' knowledge, no study has been completed with SARS-CoV-2 suspended in artificial saliva, with added protein and mucin to mimic that of human nasopharyngeal secretions.
The presence of additional salts and protein around the virus have previously been shown to have a protective effect on agents exposed to gaseous decontamination leading to incomplete inactivation of the agent [8]. This is particularly important in areas where decontamination processes are chosen for their rapid turnaround time and a more intensive exposure cycle (concentration or time) would be necessary to inactivate the agent.
This current study was the first to investigate the inactivation kinetics, taking sampling time-points, finding inactivation of SARS-CoV-2 to below the detection limit within 5 min of the injection of VHP to the enclosure. It is expected that an enveloped virus would be inactivated quickly as these are generally most easily inactivated by chemical disinfection. However, this study demonstrates that even when additional protective protein and salt media are present on non-porous surfaces, SARS-CoV-2 is rapidly inactivated by VHP.
Acknowledgements
We thank Professor K. Richards, High Containment Microbiology, PHE, Porton Down, for producing the viral stocks for this study.
Conflict of interest statement
None declared. The views expressed in this article are those of the authors and are not necessarily those of UKHSA or the Department of Health and Social Care.
Funding sources
This work was funded by MRC award MC_PC_19064 COVID-19, Understanding Environmental and Airborne Routes of Transmission.
References
- 1.World Health Organization . 2020. Transmission of SARS-CoV-2: implications for infection prevention precautions; p. 10. [Google Scholar]
- 2.Kormuth K.A., Lin K., Prussin A.J., 2nd, Vejerano E.P., Tiwari A.J., Cox S.S., et al. Influenza virus infectivity is retained in aerosols and droplets independent of relative humidity. J Infect Dis. 2018;218:739–747. doi: 10.1093/infdis/jiy221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Fischer R.J., Morris D.H., van Doremalen N., Sarchette S., Matson M.J., Bushmaker T., et al. Effectiveness of N95 respirator decontamination and reuse against SARS-CoV-2 virus. Emerg Infect Dis. 2020;26(9) doi: 10.3201/eid2609.201524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Schwartz A., Stiegel M., Greeson N., Vogel A., Thomann W., Brown M., et al. Decontamination and reuse of N95 respirators with hydrogen peroxide vapor to address worldwide personal protective equipment shortages during the SARS-CoV-2 (COVID-19) pandemic. Appl Biosaf. 2020;25:67–70. doi: 10.1177/1535676020919932. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Paton S., Spencer A., Garratt I., Thompson K.-A., Dinesh I., Aranega-Bou P., et al. Persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and viral RNA in relation to surface type and contamination concentration. Appl Environ Microbiol. 2021;87 doi: 10.1128/AEM.00526-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Falaise C., Bouvattier C., Larigauderie G., Lafontaine V., Berchebru L., Marangon A., et al. Hydrogen peroxide vapor decontamination of hazard group 3 bacteria and viruses in a biosafety level-3 laboratory. Appl Biosaf. 2021 Sep 8 doi: 10.1089/apb.2021.0022. [online ahead of print] [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Christie-Holmes N., Tyli R., Budylowski P., Guvenc F., Weiner A., Poon B., et al. Vapourized hydrogen peroxide decontamination in a hospital setting inactivates SARS-CoV-2 and HCoV-229E without compromising filtration efficiency of unexpired N95 respirators. Am J Infect Control. 2021;49:1227–1231. doi: 10.1016/j.ajic.2021.07.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Otter J.A., Yezli S., French G.L. Impact of the suspending medium on susceptibility of meticillin-resistant Staphylococcus aureus to hydrogen peroxide vapour decontamination. J Hosp Infect. 2012;82:213–215. doi: 10.1016/j.jhin.2012.08.006. [DOI] [PubMed] [Google Scholar]
