key: cord-1009689-6bxgkgjr
authors: Sallustio, Fabio; Cardinale, Gaetano; Voccola, Serena; Picerno, Angela; Porcaro, Piercarmine; Gesualdo, Loreto
title: Ozone eliminates Novel Coronavirus Sars-CoV-2 in mucosal samples
date: 2021-07-24
journal: New Microbes New Infect
DOI: 10.1016/j.nmni.2021.100927
sha: 6a787ba4bcda73a89dac3349c0009418ddd1db31
doc_id: 1009689
cord_uid: 6bxgkgjr

Recent investigations have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is able to resist on the surfaces and that the diffusion occurs through droplets which can remain suspended in the air as an aerosol. The ozone generated in situ from oxygen is an active ingredient with a "biocidal" action, but little is known about its capacity to inactivate specifically SARS-CoV-2. Here we show, for the first time, the efficiency of the ozone treatment to neutralize the SARS-CoV-2 present in nasopharynx secretion samples with high viral load. Our data show that ozone is effectiveness in SARS-CoV-2 elimination.

In early January 2020, a novel Coronavirus was identified as infective agent responsible of an 26 outbreak of viral pneumonia in Wuhan, China [1] . 27 SARS-CoV-2 is spread by human-to-human transmission: via respiratory droplets, through 28 the saliva and aerosol secretions of the upper airways carried by cough and / or sneezing [2] ; 29 direct contact, with a handshake and touching the mucous membranes of the mouth, nose and 30 eyes with contaminated hands; fecal-oral transmission [3] . 31

While it is established that aerosol entry can take place not only through the airways but also 32 through the conjunctival and oral mucous membranes, it is not clear whether a person can 33 become infected even by touching surfaces or objects contaminated by the virus and then 34 touching his mouth, nose and eyes. 35

Once expelled from the body, the virus, if contained in an aerosol, can remain suspended for 36 a time that can vary depending on the size of the liquid or solid particles to which it may be 37 attached. When deposited on different types of surfaces, it has a variable activity depending 38 on the material it encounters, but the duration of its virulence is still under study [4] . 39

For these reasons, it is important establish a valid method to sanitize the workplace and the 40 living rooms. Ozone generated in situ from oxygen is an active ingredient with a "biocidal" 41 action as a disinfectant for surfaces and drinking water and for use in cooling towers of 42 industrial plants. Ozone has a microbicidal efficacy also on viruses [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . 43

Ozone (O3) is unstable because gas degrades rapidly on its stable status, diatomic oxygen 44 (O2) with formation of atoms of free oxygen or free radical. Atoms of free oxygen or radicals 45 are highly reactive and they oxidize almost all (included virus, bacteria, organic and 46 inorganic compounds) in contacts, making ozone a potent disinfectant and oxidant. 47 with COVID-19, and these trials showed that this therapy has given benefits on patients, as 49 reducing tissue hypoxia, decreasing hypercoagulability, renal and heart protection, 50 modulating immune function, improving phagocytic function, and impairing viral replication. 51

[15-18] Studies showed that ozone exposure eliminated heat-inactivated SARS-CoV-2 in 52 different Personal Protective Equipment (PPE) components under appropriate exposure 53 times, ozone concentrations, and relative humidity conditions decreasing the risk of 54 contamination associated with personal protective equipment management and in increasing 55 its availability [19] However, little is known about ozone capacity to inactivate specifically 56 SARS-CoV-2. Here we show that ozone can inactivate the SARS-CoV-2 present in 57 secretions from nasopharynx. 58

Nasopharyngeal swabs were collected in duplicate from the 25 subjects. We collected, with 61 the same swab, both the secretions from nasopharynx and from posterior pharyngeal wall 62 and tonsillar pillars. 63

The study was carried out in accordance with the Helsinki Declaration and the European 64

Guidelines for Good Clinical Practice and written consent was obtained from all subjects. 65

The presence or absence of SARS-CoV-2 was detected by the SARS-CoV-2 Real Time kit 66 Table S1 and Supplemental 93 Table S2 ). 94

Instead, after the ozone treatment, all 25 samples resulted negative for the virus, also samples 95 with a high viral load, and no gene amplification for none of the three SARS-CoV-2 genes 96 J o u r n a l P r e -p r o o f was detectable (Figure 1 and Supplemental Table S3 ). These results showed, for the first 97 time, the efficiency of ozone treatment to neutralize the SARS-CoV-2. Its diffusion occurs 98 through Flügge's micro-drops (droplets) and it is able to remain suspended in the air as an 99 aerosol (core droplets). Flügge droplets that have a diameter > 5 microns can spread up to 1m 100 away [20] . The nuclei of the Flügge droplets (droplets nuclei) which have a diameter < 101 5micron, determine an aerosol that has a diffusion capacity of greater than 1 m [20] . 102

Ozone is able to degrade rapidly organic compounds; at higher concentrations, ozone inactive 103 quickly a wide range of pathogen (bacteria, include spores, virus, protozoa) [5, [9] [10] [11] . 104 105 Referring specifically to effectiveness against SARS-CoV-2, considering the mechanism of 106 action of ozone, a disinfectant action is conceivable. Nevertheless, at moment there are not 107 direct demonstrations about efficiency obtained in controlled studies. This is one of the first 108 studies that showed the effectiveness of ozone. Indeed, ozone is a stronger oxidant than the 109 other common disinfectants, as chlorine and hypochlorite [21] . The use of chlorine or 110 hypochlorite in many states has been reduced for the possibility of formation of cancerogenic 111 products as trihalomethanes during disinfection. On the contrary, disinfection with ozone 112 does not produce detrimental residues and all residues ozone will be converted in oxygen in 113 short time [21] . Ozone is considered an ecological disinfectant [17] . 114 115 Moreover, ozone generators are currently promoted as dispositive usable for disinfection of 116 work environments and living rooms. However, the operating conditions must be carefully 117 selected since the effectiveness of the ozonation processes varies significantly depending on 118 the characteristics of the environment to be sanitized [22] . 119

Anyway, before to use ozone to treatment locations, it is necessary to value the risk of 120 exposition as for the operator engaged for disinfection operation, as for the staff that uses the 121 equipment [22] . 123

In conclusion, here we showed that the ozone can inactivate the SARS-CoV-2 present in 124 secretions from nasopharynx, neutralizing the ORF1ab, the protein E and the N protein genes 125 and our data support the effectiveness of SARS-CoV-2 ozone disinfection in work 126 environments and living rooms. However, the ozone may have an effect on SARS-CoV-2 127 also in vivo, therefore further experiments will be needed to study in vitro and in vivo the 128 ozone effect on the COVID-19. Moreover, since ground-level ozone concentrations peaked in 129 summer [23, 24] , it is conceivable that ozone may contribute to the low levels of SARS-CoV-130 2 circulating in the warmer months. 131 132 133

Effects of Ozone Treatment on Personal Protective 188 Equipment Contaminated with SARS-CoV-2

The Lancet Respiratory Medicine. COVID-19 transmission-up in the air

Consequences of chemical impact of disinfectants: safe 193 preventive measures against COVID-19

Effective Use of Ozone as Air and Surface Disinfectant in the Conjuncture of Covid-197 19

Diurnal, seasonal and weekdays-weekends variations of ground level 199 ozone concentrations in an urban area in greater Cairo

Seasonal characteristics of ambient nitrogen oxides 202 and ground-level ozone in metropolitan northeastern New Jersey

Figure 1. The graphs represent the Real-Time PCR results (Ct) of nasopharyngeal swab with 211 samples before (color blue) and after treatment (orange) with ozone for the three genes of 212

SARS-CoV-2: ROX for RdRp and RdRp/Hel genus of the regions Orf1ab

FAM for E region