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Citation: Torres-Mata, L.B.;
García-Pérez, O.; Rodríguez-
Esparragón, F.; Blanco, A.; Villar, J.;
Ruiz-Apodaca, F.; Martín-Barrasa,
J.L.; González-Martín, J.M.;
Serrano-Aguilar, P.; Piñero, J.E.; et al.
Ozone Eliminates SARS-CoV-2 from
Difficult-to-Clean Office Supplies and
Clinical Equipment. Int. J. Environ.
Res. Public Health 2022,19, 8672.
https://doi.org/10.3390/
ijerph19148672
Academic Editor: Giuseppe La Torre
Received: 14 June 2022
Accepted: 14 July 2022
Published: 16 July 2022
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International Journal of
Environmental Research
and Public Health
Article
Ozone Eliminates SARS-CoV-2 from Difficult-to-Clean Office
Supplies and Clinical Equipment
Laura B. Torres-Mata 1,2,3,4 , Omar García-Pérez 5,6,7, Francisco Rodríguez-Esparragón1,2,5,8 , Angeles Blanco 4,
Jesús Villar 1,9 ,10 , Fernando Ruiz-Apodaca 11, JoséL. Martín-Barrasa 1,2,8,12 , Jesús M. González-Martín1,2,5,9,
Pedro Serrano-Aguilar 13, 14,15 , JoséE. Piñero 5,7,8,16 , Elizabeth Córdoba-Lanús5,6,7,8 ,
Jacob Lorenzo-Morales 5,7,8,16,* and Bernardino Clavo 1,2,3,5,8,17,18,*
1Research Unit, Hospital Universitario Dr. Negrín, 35019 Las Palmas de Gran Canaria, Spain;
lbtm1002@gmail.com (L.B.T.-M.); frodesp@gobiernodecanarias.org (F.R.-E.); jesus.villar54@gmail.com (J.V.);
jmarbars@gobiernodecanarias.org (J.L.M.-B.); josu.estadistica@gmail.com (J.M.G.-M.)
2Fundación Canaria del Instituto de Investigación Sanitaria de Canarias (FIISC),
35019 Las Palmas de Gran Canaria, Spain
3BioPharm Group, Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS),
Universidad de Las Palmas de Gran Canaria, 35016 Las Palmas de Gran Canaria, Spain
4Chemical Engineering & Materials Department, Universidad Complutense, 28040 Madrid, Spain;
ablanco@ucm.es
5
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna,
Tenerife, 38200 La Laguna, Spain; omargp6@gmail.com (O.G.-P.); jpinero@ull.edu.es (J.E.P.);
acordoba@ull.edu.es (E.C.-L.)
6Departamento de Medicina Interna, Dermatología y Psiquiatría, Universidad de La Laguna,
Tenerife, 38200 La Laguna, Spain
7Red Cooperativa de Enfermedades Tropicales (RICET), Instituto de Salud Carlos III, 28029 Madrid, Spain
8CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, 28029 Madrid, Spain
9CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, 28029 Madrid, Spain
10 Li Ka Shing Knowledge Institute at St Michael’s Hospital, Toronto, ON M5B 1T8, Canada
11 Lighting Dynamic Technology, SL, 35001 Las Palmas, Spain; fernando.ruiz@hispalux.com
12
Aquaculture and Wild Species Health, Infectious Diseases, Universitary Institute of Animal Health and Food
Safety (IUSA), Universidad de Las Palmas de Gran Canaria, 35413 Arucas, Spain
13 Red de Investigación en Cronicidad, Atención Primaria y Promoción de la Salud (RICAPPS),
Instituto de Salud Carlos III, 28029 Madrid, Spain; pseragu@gobiernodecanarias.org
14 Servicio de Evaluación y Planificación del Servicio Canario de Salud (SESCS),
38109 Santa Cruz de Tenerife, Spain
15 Red de Agencias de Evaluación de Tecnologías Sanitarias y Prestaciones del Sistema Nacional de
Salud (RedETS), 28071 Madrid, Spain
16 Departamento de Obstetricia, Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología,
Medicina Legal y Forense y Parasitología, Universidad de La Laguna, Tenerife, 38200 La Laguna, Spain
17 Chronic Pain Unit, Hospital Universitario Dr. Negrín, 35019 Las Palmas de Gran Canaria, Spain
18
Radiation Oncology Department, Hospital Universitario Dr. Negrín, 35019 Las Palmas de Gran Canaria, Spain
*Correspondence: jmlorenz@ull.edu.es (J.L.-M.); bernardinoclavo@gmail.com (B.C.)
Abstract:
(1) Background: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)
continues to cause profound health, economic, and social problems worldwide. The management
and disinfection of materials used daily in health centers and common working environments have
prompted concerns about the control of coronavirus disease 2019 (COVID-19) infection risk. Ozone
is a powerful oxidizing agent that has been widely used in disinfection processes for decades. The
aim of this study was to assess the optimal conditions of ozone treatment for the elimination of heat-
inactivated SARS-CoV-2 from office supplies (personal computer monitors, keyboards, and computer
mice) and clinical equipment (continuous positive airway pressure tubes and personal protective
equipment) that are difficult to clean. (2) Methods: The office supplies and clinical equipment were
contaminated in an area of 1 cm
2
with 1
×
10
4
viral units of a heat-inactivated SARS-CoV-2 strain,
then treated with ozone using two different ozone devices: a specifically designed ozonation chamber
(for low–medium ozone concentrations over large volumes) and a clinical ozone generator (for
high ozone concentrations over small volumes). SARS-CoV-2 gene detection was carried out using
quantitative real-time polymerase chain reaction (RT-qPCR). (3) Results: At high ozone concentrations
Int. J. Environ. Res. Public Health 2022,19, 8672. https://doi.org/10.3390/ijerph19148672 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2022,19, 8672 2 of 13
over small surfaces, the ozone eliminated SARS-CoV-2 RNA in short time periods—i.e., 10 min (at
4000 ppm) or less. The optimum ozone concentration over large volumes was 90 ppm for 120 min
in ambient conditions (24
◦
C and 60–75% relative humidity). (4) Conclusions: This study showed
that the appropriate ozone concentration and exposure time eliminated heat-inactivated SARS-CoV-2
RNA from the surfaces of different widely used clinical and office supplies, decreasing their risk of
transmission, and improving their reutilization. Ozone may provide an additional tool to control the
spread of the COVID-19 pandemic.
Keywords: COVID-19; SARS-CoV-2; surface disinfection; clinical equipment; office supplies; ozone
1. Introduction
The World Health Organization (WHO) declared severe acute respiratory syndrome
coronavirus type 2 (SARS-CoV-2) a pandemic on 11 March 2020, and almost two years
later, it is still causing profound health, economic, and social problems worldwide [
1
]. The
persistence of SARS-CoV-2 on environmental surfaces has been considered a potentially
critical factor for viral spread, despite conflicting reports regarding the maintenance of
SARS-CoV-2 infectivity on different surfaces [2–5].
SARS-CoV-2 belongs to the Betacoronavirus family and the group IV Nidovirals order
(Baltimore classification). This group includes positive monocatenary RNA viruses. Its
genome contains 80% similarity to SARS-CoV-1; both have a capsid, which confers sensi-
tivity to heating, detergents, and solvents. The survival of SARS-CoV-2 is dependent on
the environmental temperature, relative humidity, and pH, as well as the reactivity of the
surface where is located. It is viable on unanimated surfaces for prolonged periods of time
between 2 h and nine days, and is spontaneously inactivated on copper surfaces between
4h and 8 h, at 24 h on cardboard, at 48 h on stainless steel, and at 72 h on plastics [
3
,
4
,
6
,
7
].
Recently, our group reported extended survival times of five to seven days, assessed by
evaluation of its cytopathic effect in VERO cells (kidney epithelial cells extracted from an
African green monkey), and by gene detection in face masks up to 30 days after contamina-
tion [
5
]. Although vaccinations are the top priority for reducing the spread of coronavirus
disease 2019 (COVID-19), according to the WHO guidelines the effective prevention and
control of infection includes a practical, evidence-based approach to resist disease spread-
ing [
8
]. Therefore, the effective disinfection of clinical and public materials may play a
crucial role in limiting the viral spread and accelerating the reuse of these materials. Within
this context, ozone has been a subject of growing interest [9,10]
Ozone (O
3
) is a molecular gas with three oxygen atoms bonded by high-energy co-
valent bonds, which makes ozone a powerful oxidizing agent and, therefore, a highly
antimicrobial agent. Although it is mainly used for water treatment, it has also been
proven to be highly effective at eliminating bacteria, fungi, and molds, and inactivating
viruses, including the SARS virus, on surfaces and in aerosols suspended in the
air [11–15]
.
Its efficiency depends on the treatment conditions (e.g., concentration, exposure time,
temperature, and humidity) and material properties (e.g., surface reactivity and poros-
ity) [
7
,
10
,
11
,
16
–
18
]. Its high oxidant activity affects polyunsaturated acids in the biological
membranes of bacteria, molds, fungi, and viruses, while also oxidizing nucleic acids (DNA
and RNA) [
19
,
20
]. In addition to its wide microbicide spectrum activity, ozone does not
generate reaction by-products due to its rapid decomposition into oxygen in the atmosphere
(see equation in Supplemental Material for further details) [21].
The European Union has included ozone as an effective biocide for water waste
cleansing (EU Biocidal Products Regulation N
º
528/2012). Due to the physical state of
ozone, it tends to expand and occupy the entire volume in which it is contained, which
is an advantage when compared to other disinfection systems, such as ultraviolet (UV)
irradiation or hypochlorite dissolutions [17,22–24].
Int. J. Environ. Res. Public Health 2022,19, 8672 3 of 13
Ozone can be generated by a diverse set of devices that use electrical corona discharge
to produce ozone from oxygen in the air, or that use medical-grade oxygen. In recent years,
there has been practically no innovation in the development of ozone generators in industry,
due to the low demand for these devices. However, due to the COVID-19 pandemic, the
variety, applicability, and versatility of ozone generators are growing quickly [
14
,
15
,
25
–
27
].
Currently, multiple ongoing studies are attempting to optimize the inactivation and/or
elimination of SARS-CoV-2 using ozone [
10
,
11
,
14
,
15
]. It has been reported that ozone
treatment is a widely accessible and effective method for the disinfection of several materials
from SARS-CoV-2, including personal protective equipment (PPE) for healthcare workers
and patients [
10
,
22
,
25
], creating the possibility of safely re-using these clinical and working
materials and contributing to their more sustainable use. However, further studies are still
necessary to validate these results and facilitate the wide acceptance of this treatment and
its inclusion in the list of viable treatments.
In our previous work, we reported the RNA degradation of heat-inactivated SARS-
CoV-2 on PPE and masks at high and low ozone concentrations [
10
]. The aim of the
current work was to validate these preliminary results with further materials. Thus, this
study was extended to the contaminated surfaces of different office supplies and clinical
equipment that are difficult to clean, where the effects of ozone treatment were evaluated
for eliminating heat-inactivated SARS-CoV-2 RNA.
2. Methods
2.1. Samples, Study Design, and Outcome Assessment
Samples of several materials and sizes were contaminated with heat-inactivated
SARS-CoV-2
. The size of the samples varied from bands of 2 cm
×
1 cm (for face masks, and
vinyl and nitrile lab gloves) to the entire object in the case of office, clinical, and laboratory
supplies (up to 40 cm
×
40 cm
×
20 cm), including two operative cellphones, inoperative
computer accessories (mouse, keyboard, and computer screen), reactant flasks, test tubes,
grids, continuous positive airway pressure (CPAP) tubes (100 cm length
×
2 cm diameter),
and syringe and needle covers (paper tissue).
All the supplies assessed were contaminated with the SARS-CoV-2 strain 2019-nCoV/
USA-WA1/2020, which was inactivated by heating at 65
◦
C for 30 min (ATCC
®
VR-
1986HK
™
, ATCC, Manassas, VA, USA) at 1
×
10
3
copies/
µ
L. In all cases, the volume of the
contamination drop was 10
µ
L (occupying a surface equivalent to 1 cm
2
), corresponding
to 1
×
10
4
copies, which was considered a reasonable amount of virus to remain stable
on a surface for enough time to experimentally evaluate the virucidal activity of the
procedure [
7
,
13
,
28
]. After that, the samples were allowed to dry in a laminar flow hood
until treatment with the corresponding ozone conditions.
For the first assay, we used low concentrations of ozone (19 ppm) under high relative
humidity (80–95%) to reinforce the ozone effect at low concentrations, as previously re-
ported [
7
,
10
,
11
,
28
–
30
]. In the following eight assays, the temperature (21.8–24.7
◦
C) and
relative humidity (60–75%) were those of room conditions, controlled by the integral air
conditioning system of the hospital.
For each study performed on every supply type, two samples were used for the control
(confirmation “pre-treatment” column) and another two samples were used for the O
3
treatment (“post-treatment” column).
The primary outcome measure was the detection yield of heat-inactivated SARS-CoV-2
gene amplification after ozone treatment, as assessed by quantitative real-time polymerase
chain reaction (RT-qPCR).
Table 1shows the different materials and supplies assessed in the different ozone
treatment conditions.
Int. J. Environ. Res. Public Health 2022,19, 8672 4 of 13
Table 1.
SARS-CoV-2 gene amplification by quantitative real-time polymerase chain reaction (RT-
qPCR) in clinical and office supplies contaminated by a heat-inactivated strain, after treatment with
different ozone exposure conditions (concentration, time, and relative humidity).
Supply Type
(by Duplicate)
Ozone Concentration
(ppm) Time of Treatment
(Minutes)
Relative Humidity
(%)
RT-qPCR
Pre-Treatment Post-Treatment
Face masks
19 30 80–90 XXX XX
19 60 85–90 XXX X
90 120 65–70 XXX X
2000 5 60–75 XXX X
2000 10 60–75 XXX X
4000 5 60–75 XXX X
Vinyl lab glove 19 30 80–90 XXX X
Nitrile lab glove 90 120 65–70 XXX X
Cover needle 90 120 65–70 XXX X
Cover syringe 90 120 65–70 X n.v.
CPAP tube
70 a120 60–75 XXX X
90 a120 65–70 XXX X
4000 b10 60–75 XXX X
10,000 b10 60–75 XXX X
Lab grid 70 120 60–75 XXX X
90 120 65–70 XXX X
Reactant flask 70 120 60–75 XXX X
90 120 65–70 XXX X
Reactant flask tag 70 120 60–75 X n.v.
90 120 65–70 X n.v.
Test tube 70 120 60–75 XXX X
90 120 65–70 XXX X
Between keys
of mouse
70 120 60–75 X n.v.
90 120 65–70 X n.v.
Computer mouse
33 120 60–75 XXX X
70 120 60–75 XXX XXX
90 120 65–70 XXX X
4000 10 60–75 XX
10,000 10 60–75 XX
Computer screen
33 120 60–75 XXX XXX
70 120 60–75 XXX X
90 120 65–70 XXX X
4000 10 60–75 XXX X
10,000 10 60–75 XXX X
Keyboard key
33 120 60–75 XXX XXX
70 120 60–75 XXX X
90 120 65–70 XXX X
4000 10 60–75 X n.v.
10,000 10 60–75 X n.v.
Between keys
of keyboard
33 120 60–75 XXX XXX
70 120 60–75 XXX XXX
90 120 65–70 X n.v.
4000 10 60–75 XXX X
10,000 10 60–75 XXX X
Cellphone 70 120 60–75 XXX X
90 120 65–70 XXX X
For each study performed on every supply type, two samples were used for the control (confirmation “pre-
treatment” column) and another two samples were used for the O
3
treatment (“post-treatment” column). For each
sample, RT-qPCR was performed in duplicate. X, no amplification;
X
, one positive gene;
XX
, two positive genes;
XXX
, three positive genes; CPAP, continuous positive airway pressure; n.v., not valuable due to negative result in
the control in the pretreatment group;
a
contaminating drop at 50 cm from the entry point of O
3
;
b
contaminating
drop at 1 m (100 cm) from the entry point of O3.
2.2. Ozone Exposure Conditions
The ozone treatment procedures were performed at the Hospital Universitario de
Gran Canaria Dr. Negrín (Las Palmas de Gran Canaria, Spain).
For the high-concentration assays, the ozone was produced using a medical ozone
generator (Ozonobaric P
®
, Sedecal, Madrid, Spain). This device generates ozone from
medical-grade oxygen, obtaining an O
3
/O
2
gas mixture between 500 ppm and 40,000 ppm
(1–80 g/m3)
in relatively small volumes. During the procedure, the samples contaminated
Int. J. Environ. Res. Public Health 2022,19, 8672 5 of 13
with heat-inactivated SARS-CoV-2 were introduced one at a time in a 60 mL syringe (when
possible) or inside a plastic bag. Then, the air was expelled by a vacuum. Later, an
O
3
/O
2
gas mixture was introduced at selected concentrations of
2000, 4000, or 10,000 ppm
(
4, 8, or 20 g/m3
, respectively) with short exposition times (5 or 10 min) based on our
previous study [10].
The low-concentration assays were performed with a size-adaptable ozonation cham-
ber UVOZ
®
(designed and performed by Lighting Dynamic Technology, Las Palmas
de Gran Canaria, Spain). The chamber was used with a cabinet with dimensions of
200 ×100 ×100 cm3
(2000 L) and was equipped with a 65 W industrial ozone generator,
which produced ozone from the oxygen present in the environmental air (21%). It was
also equipped with a set of two ultraviolet (UV) lamps and a humidifier to be used as
required. Using the large volume of the ozonation chamber, we analyzed the effect of
low ozone concentrations (19, 33, 70, and 90 ppm = 0.038, 0.066, 0.140, and 0.180 g/m
3
,
respectively) for longer exposure times (30, 60, 90, and 120 min). To compensate for the
spontaneous decomposition of ozone to oxygen (half-life of 40 min at 20
◦
C and 25 min
at 30
◦
C), the ozonation chamber’s ozone generator was switched on and off to maintain
the concentration at the desired values during the experiments. Table 1shows the ozone
treatment conditions.
For each supply, at every evaluated ozone condition, two units were used. For each
unit we obtained two pre-ozone control samples and two post-ozone samples, which were
collected with swabs (or by cutting the sample to pick up the drop) and maintained in
3 mL
universal transport medium (UTM-RT
™
, COPAN Diagnostics, CA) for conservation
and further PCR analyses. Each sample was assessed in duplicate by RT-qPCR for the
amplification of SARS-CoV-2 genes.
2.3. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)
Detection of viral RNA by RT-qPCR was performed at the Instituto Universitario
de Enfermedades Tropicales y Salud Pública de Canarias (La Laguna, Tenerife, Spain)
according to the WHO guidelines for Biosafety Level 2 facilities [
31
]. The previously
described samples were processed to extract viral RNA using a Maxwell 16S Viral RNA
Mini Kit (Promega, Madrid, Spain) following the manufacturer’s recommendations. The
extracted RNA was resuspended in 50 µL of elution buffer and used for RT-qPCR.
For viral gene detection by RT-qPCR, the TaqPath
™
COVID-19 CE-IVD RT-qPCR
Kit (Applied Biosystems, Thermo Fisher Scientific, Madrid, Spain) was used, following
the manufacturer’s instructions. This kit included assays targeting three SARS-CoV-2
genes (Gene ORF1ab, N Protein, and S Protein), and an MS2 Phage as a control for the
RNA extraction. It also contained a positive TaqPath
™
COVID-19 control. Each sample
was analyzed in duplicate with a QuantStudio 3
™
Real-Time qPCR System (Applied
Biosystems). All RT-qPCR samples were assessed in duplicate. Positive results were
considered when amplification genes had Ct values <37 (Ct: Cycle threshold related to the
number of cycles required for the fluorescently marked amplification to cross the threshold
in the RT-qPCR reaction).
3. Results
3.1. Low Ozone Concentrations
At 19 ppm (0.038 g/m
3
) and controlled relative humidity (80–90%), viral gene ampli-
fication was detected in face masks after 30 min (two genes) and 60 min (only one gene)
of ozone treatment. For the vinyl lab gloves, no amplification was observed after 30 min
of treatment.
At 33 ppm (0.066 g/m
3
) and standard relative humidity, only the office supplies were
studied, and viral genes were detected in all the analyzed materials (computer mouse,
computer screen, and keyboard keys).
At 70 ppm (0.140 g/m
3
) and standard relative humidity, the clinical equipment, and
office supplies were studied. The treatment was effective for six of the nine materials,
Int. J. Environ. Res. Public Health 2022,19, 8672 6 of 13
including the lab grid, reactant flask, test tube, computer screens, keyboard keys, and
cellphone screens. In the CPAP tube, the contaminated drop at 50 cm from the entry point
of O3showed amplification in only one gene.
At 90 ppm (0.180 g/m
3
) and standard relative humidity, all evaluable clinical equip-
ment and office supplies showed no SARS-CoV-2 gene amplification after ozone treatment.
Table 1shows the results of the different ozone exposure conditions. Figure 1shows
the RT-qPCR results of heat-inactivated SARS CoV-2 genes evaluated for three different
materials (face mask, cellphone, and lab grid) treated with ozone at 90 ppm for 120 min at
65–70% relative humidity.
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 7 of 13
Figure 1. Heat-inactivated SARS-CoV-2 gene evaluation by RT-qPCR for (A,B) face mask, (C,D) lab
grid, and (E,F) cellphone treated with 90 ppm of ozone for 120 min (65–70% humidity). Target MS2:
MS2 Phage as a control for the RNA extraction; Target O, N, and S: specific SARS-CoV-2 target
sequences in the ORF1ab, nucleocapsid, and spike protein gene respectively. (A,C,E) images corre-
spond to materials before exposure to ozone that showed Ct values <37, indicating positive ampli-
fication of the three viral genes. (B,D,F) images correspond to materials treated with ozone that
showed no amplification of the viral targets O, N, or S, where only the control target MS2 was am-
plified.
3.2. High Ozone Concentrations
At 2000 ppm (4 g/m
3
) and standard relative humidity, we only analyzed face masks,
and they did not show gene amplification after 10 min nor 5 min of O
3
exposition.
At 4000 ppm (8 g/m
3
) and standard relative humidity, there was no gene amplifica-
tion after 5 min in face masks, and there was no gene amplification after 10 min on the
computer mouse, computer screen, keyboard keys, nor between keys of the keyboard.
However, one gene remained amplified after 10 min in the contaminated drop at 1 m (100
cm) from the entry point of O
3
.
At 10,000 ppm (20 g/m
3
), and standard relative humidity, there was no viral RNA
detection after 10 min in any clinical equipment nor office supplies, including the areas
between keys of the keyboard, nor the contaminated drops at 1 m (100 cm) from the entry
point of O
3
. Table 1 shows the results obtained under low and high ozone concentrations.
Figure 1.
Heat-inactivated SARS-CoV-2 gene evaluation by RT-qPCR for (
A
,
B
) face mask,
(C,D) lab
grid, and (
E
,
F
) cellphone treated with 90 ppm of ozone for 120 min (65–70% humidity). Target
MS2: MS2 Phage as a control for the RNA extraction; Target O, N, and S: specific SARS-CoV-2
target sequences in the ORF1ab, nucleocapsid, and spike protein gene respectively. (
A
,
C
,
E
) images
correspond to materials before exposure to ozone that showed Ct values <37, indicating positive
amplification of the three viral genes. (
B
,
D
,
F
) images correspond to materials treated with ozone
that showed no amplification of the viral targets O, N, or S, where only the control target MS2
was amplified.
3.2. High Ozone Concentrations
At 2000 ppm (4 g/m
3
) and standard relative humidity, we only analyzed face masks,
and they did not show gene amplification after 10 min nor 5 min of O3exposition.
At 4000 ppm (8 g/m
3
) and standard relative humidity, there was no gene amplification
after 5 min in face masks, and there was no gene amplification after 10 min on the computer
Int. J. Environ. Res. Public Health 2022,19, 8672 7 of 13
mouse, computer screen, keyboard keys, nor between keys of the keyboard. However, one
gene remained amplified after 10 min in the contaminated drop at 1 m (100 cm) from the
entry point of O3.
At 10,000 ppm (20 g/m
3
), and standard relative humidity, there was no viral RNA
detection after 10 min in any clinical equipment nor office supplies, including the areas
between keys of the keyboard, nor the contaminated drops at 1 m (100 cm) from the entry
point of O3. Table 1shows the results obtained under low and high ozone concentrations.
4. Discussion
This study indicates that ozone could eliminate heat-inactivated SARS-CoV-2 genes
from different contaminated surfaces of several office and clinical supplies. This effect
was highly dependent on the ozone concentration, time exposure, and material used. At a
standard relative humidity (60–75%) and a temperature of 21.8–24.7
◦
C, we found that the
best disinfection conditions were 90 ppm for 120 min for large volume supplies. However,
for smaller volumes, 4000 ppm for 10 min was sufficient, although 10,000 ppm was required
for surfaces that were more difficult to access (i.e., 100 cm CPAP tube).
As previously reported, at low ozone concentrations, high-humidity conditions rein-
force ozone activity [
7
,
10
,
11
,
28
–
30
]. Thus, the disinfection treatment was started at an ozone
concentration of 19 ppm (0.038 g/m
3
) and 80–95% relative humidity. The samples initially
selected were vinyl lab gloves and face masks, due to the high use of these tools during the
COVID-19 pandemic. The treatment was applied to the vinyl lab gloves for 30 min, and to
the face masks for 30 and 60 min. The treatment was successful for the vinyl lab gloves,
which did not show the amplification of SARS-CoV-2 genes, but not for the face masks. The
improved results for the gloves compared to those for the face masks could be due to the
less porous surface of gloves that may allow ozone to easily access and react effectively
with the viral RNA. Although the reported survival time of SARS-CoV-2 on plastic mate-
rials is 72 h (polypropylene in masks and polyvinylchloride in gloves), their microscopic
interactions seem to influence the efficacy of the procedure [
4
]. This finding is in agreement
with our previous report showing the elimination of heat-inactivated SARS-CoV-2 from
PPE gowns at low ozone concentrations (4–6.5 ppm) under 99% relative humidity, but not
from face masks [
10
]. In the current study, we did not use 99% relative humidity, because in
previous work we found water condensation on the surfaces inside the ozonation chamber.
The 80–90% relative humidity used in the first assay did not lead to water condensation on
the surfaces inside the ozonation chamber. Finally, because the study included electronic
and personal computer (PC) components, we decided to evaluate the ozone effects under
standard relative humidity, which is more tolerable for many electronic devices.
Inside the ozonation chamber after 120 min of ozone exposure, we observed that:
(i) at an ozone concentration of 33 ppm (0.066 g/m
3
), SARS-CoV-2 RNA maintained its
integrity, as observed by the amplification of viral genes; (ii) at a concentration of 70 ppm
(0.140 g/m3)
, ozone was effective on six out of nine samples; and (iii) at a concentration
of 90 ppm (0.180 g/m
3
), ozone was effective in eliminating SARS-CoV-2 RNA from all
the tested surfaces. This work also assessed the efficacy of the use of very high ozone
concentrations with short exposure time for degrading the RNA of SARS-CoV-2. Ozone
treatments at 4000 ppm (8 g/m
3
) and 2000 ppm (4 g/m
3
) for five minutes were effective for
face masks, in agreement with previous reports [
10
,
32
], whilst ozone exposure at
4000 ppm
for 10 min was effective for all surfaces, except the 100 cm CPAP tube (where one of the
three SARS-CoV-2 genes was detected); no SARS-CoV-2 genes were detected in any sample
(100 cm CPAP tube included) after 10 min at 10,000 ppm (20 g/m3).
According to previous reports, the ozone levels required to inactivate viral particles
are quite low compared to those evaluated in this study for viral RNA elimination [
11
].
This could be due to the role of envelope integrity in maintaining the infectivity of the
viral particle, and the relatively lower reactivity of RNA to oxidation compared to other
biomolecules. Lipids, by peroxidation, and proteins of the viral capsid, by losing their
tridimensional structure, affect the infectious capacity of the virus [
19
,
27
], although RNA
Int. J. Environ. Res. Public Health 2022,19, 8672 8 of 13
can persist and be detected by RT-qPCR (Figure 2). The inactivation of several viruses
through protein shell damage and lipid envelope peroxidation by ozone has been previously
reported [
19
,
20
]. A recent report described three main mechanisms for the elimination
of SARS-CoV-2 by ozone: (i) the peroxidation of unsaturated fatty acids, which leads to
the disturbance of the viral envelope formation; (ii) unsettling of the amino acid structure,
which collaborates with the viral envelope damage and leads to the oxidation of cysteine
to cystine; and (iii) the latter alongside the release of Zn
+2
from the viral non-structural
proteins, leading to secondary and tertiary structure alterations in those non-structural
proteins [33].
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 9 of 13
structural proteins, leading to secondary and tertiary structure alterations in those non-
structural proteins [33].
Figure 2. Proposed degradation effects of ozone on the biomolecules of a SARS-CoV-2 viral particle.
(A) Low ozone concentrations or short exposure times mainly lead to degradation of lipids and
proteins but not viral RNA, which is detected by RT-qPCR. (B) High ozone concentrations or long
exposure times also alter and oxidize the viral RNA, which therefore is not detected by RT-qPCR.
When assessing the paper samples, including syringe covers, reactant flask tags, and
needle covers, we observed a particular behavior. We expected gene amplification in all
samples before ozone treatment; however, there viral amplification was observed only in
the pre-ozone needle cover samples, with no amplification in the pre-ozone syringe cover
or reactant flask tag samples. Thus, the absence of amplification in the post-ozone paper
samples was not valuable. In previous viability studies of SARS-CoV-1 and SARS-CoV-2
on several materials, the data were reported as “noisier” for experiments using cardboard
or cotton (which are made of cellulose), both of which have a high adsorption capacity
[3,34,35]. This finding is likely due to the interactions between those biomaterials and the
viral particles caused by the intermolecular forces (i.e., hydrogen bonds) between RNA
hydroxyl groups and cellulose polar groups. The use of cellulose columns to purify nu-
cleic acids supports this hypothesis [36–38]. See Figure 3.
These interactions are thought to lead to the “adsorption” of RNA, which will not be
detected by RT-qPCR. The stability and interactions of SARS-CoV-2 RNA with highly po-
lar and porous materials, such as paper and cardboard, should be further studied.
On the other hand, the survival of SARS-CoV-2 on unanimated surfaces has been
described as between 4 and 8 h on copper surfaces, 24 h on cardboard, 48 h on stainless
steel, and 72 h on plastics [2,3,7]. The long-term survival of SARS-CoV-2 on plastics (a
widely used material) is a risk for contamination and propagation among people [35].
Recent data from our group found SARS-CoV-2 RNA on the surface of contaminated face
masks after 30 days [5]. Sodium hypochlorite (bleach) is the standard method for cleaning
at-risk surfaces. However, not all materials can be treated with this method or with liq-
uids, as is the case for face masks, some PPE, paper or cardboard packaging (e.g., syringe
covers, needle covers, reactant flask tags, etc.), electronic devices, and some potentially
reusable materials such as CPAP tubes.
Figure 2.
Proposed degradation effects of ozone on the biomolecules of a SARS-CoV-2 viral particle.
(
A
) Low ozone concentrations or short exposure times mainly lead to degradation of lipids and
proteins but not viral RNA, which is detected by RT-qPCR. (
B
) High ozone concentrations or long
exposure times also alter and oxidize the viral RNA, which therefore is not detected by RT-qPCR.
When assessing the paper samples, including syringe covers, reactant flask tags, and
needle covers, we observed a particular behavior. We expected gene amplification in all
samples before ozone treatment; however, there viral amplification was observed only
in the pre-ozone needle cover samples, with no amplification in the pre-ozone syringe
cover or reactant flask tag samples. Thus, the absence of amplification in the post-ozone
paper samples was not valuable. In previous viability studies of SARS-CoV-1 and SARS-
CoV-2 on several materials, the data were reported as “noisier” for experiments using
cardboard or cotton (which are made of cellulose), both of which have a high adsorption
capacity [
3
,
34
,
35
]. This finding is likely due to the interactions between those biomaterials
and the viral particles caused by the intermolecular forces (i.e., hydrogen bonds) between
RNA hydroxyl groups and cellulose polar groups. The use of cellulose columns to purify
nucleic acids supports this hypothesis [36–38]. See Figure 3.
These interactions are thought to lead to the “adsorption” of RNA, which will not be
detected by RT-qPCR. The stability and interactions of SARS-CoV-2 RNA with highly polar
and porous materials, such as paper and cardboard, should be further studied.
On the other hand, the survival of SARS-CoV-2 on unanimated surfaces has been
described as between 4 and 8 h on copper surfaces, 24 h on cardboard, 48 h on stainless steel,
and 72 h on plastics [
2
,
3
,
7
]. The long-term survival of SARS-CoV-2 on plastics (a widely
used material) is a risk for contamination and propagation among people [
35
]. Recent
data from our group found SARS-CoV-2 RNA on the surface of contaminated face masks
after 30 days [
5
]. Sodium hypochlorite (bleach) is the standard method for cleaning at-risk
surfaces. However, not all materials can be treated with this method or with liquids, as
is the case for face masks, some PPE, paper or cardboard packaging (e.g., syringe covers,
needle covers, reactant flask tags, etc.), electronic devices, and some potentially reusable
materials such as CPAP tubes.
Int. J. Environ. Res. Public Health 2022,19, 8672 9 of 13
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 10 of 13
Figure 3. Representation of suggested hydrogen bond interactions between cellulose and RNA. HB,
hydrogen bond.
Our findings support the effectiveness of ozone treatment for degrading SARS-CoV-
2 RNA on the surface of several difficult-to-clean supplies from clinical and office envi-
ronments that cannot be thoroughly cleaned using sodium hypochlorite. The treatment of
these supplies with ozone could represent a safe approach to prevent or decrease the risk
of contamination with SARS-CoV-2 in hospitals, nursing homes, and more general envi-
ronments. The use of ozonation chambers can facilitate the simultaneous treatment of
large or multiple devices and supplies, potentially including full PPE, instead of using
lower ozone concentrations with longer exposure times. Furthermore, the treatments can
be performed in small rooms. If faster disinfection is required (for example, for fast reuti-
lization), smaller volumes with higher ozone concentrations from clinical ozone devices
could be used for 5–10 min. Both procedures were completely safe for the operators. A n
operational strength of the ozonation chambers is the versatility of their application. They
allow treatment of different types and sizes of materials and the modulation of relative
humidity.
Our results support the potential use of ozone for the re-utilization of certain materi-
als under conditions of very low availability. Additionally, ozone does not generate con-
taminating decomposition subproducts (O
3
spontaneously degrades to O
2
), which could
decrease biological risk in the management or elimination of hazardous materials. This
can facilitate the re-utilization of supplies, decreasing waste materials, which is in line
with the green chemistry technologies associated with the Green Deal Goal of the Euro-
pean Union: preserving our environment [39].
We acknowledge some limitations of our study. First, the analyses of the computer
mouse and keyboard samples and the paper surface samples (reactant flask tag and sy-
ringe cover) were not evaluable, because the pre-ozone control samples did not show viral
Figure 3.
Representation of suggested hydrogen bond interactions between cellulose and RNA. HB,
hydrogen bond.
Our findings support the effectiveness of ozone treatment for degrading SARS-CoV-2
RNA on the surface of several difficult-to-clean supplies from clinical and office environ-
ments that cannot be thoroughly cleaned using sodium hypochlorite. The treatment of
these supplies with ozone could represent a safe approach to prevent or decrease the risk
of contamination with SARS-CoV-2 in hospitals, nursing homes, and more general environ-
ments. The use of ozonation chambers can facilitate the simultaneous treatment of large or
multiple devices and supplies, potentially including full PPE, instead of using lower ozone
concentrations with longer exposure times. Furthermore, the treatments can be performed
in small rooms. If faster disinfection is required (for example, for fast reutilization), smaller
volumes with higher ozone concentrations from clinical ozone devices could be used for
5–10 min. Both procedures were completely safe for the operators. A n operational strength
of the ozonation chambers is the versatility of their application. They allow treatment of
different types and sizes of materials and the modulation of relative humidity.
Our results support the potential use of ozone for the re-utilization of certain ma-
terials under conditions of very low availability. Additionally, ozone does not generate
contaminating decomposition subproducts (O
3
spontaneously degrades to O
2
), which
could decrease biological risk in the management or elimination of hazardous materials.
This can facilitate the re-utilization of supplies, decreasing waste materials, which is in line
with the green chemistry technologies associated with the Green Deal Goal of the European
Union: preserving our environment [39].
Int. J. Environ. Res. Public Health 2022,19, 8672 10 of 13
We acknowledge some limitations of our study. First, the analyses of the computer
mouse and keyboard samples and the paper surface samples (reactant flask tag and syringe
cover) were not evaluable, because the pre-ozone control samples did not show viral gene
amplification. After the planned analysis of two units for each supply and two samples for
each unit produced the same results, we decided not to perform further studies. Second,
nitrile and latex react very easily with ozone and were excluded from ozone treatment [
40
].
Third, the PC components were inoperative when treated, and it was not possible to
evaluate the potential adverse effect of ozone on their functionality. However, working
cellphones remained operative after ozone treatment, and the quality of the materials was
not macroscopically affected. Further evaluation is required of the effects on these materials
of chronic exposure to ozone, especially electronic components.
5. Conclusions
This study shows that an appropriate ozone concentration and exposure time can elim-
inate heat-inactivated SARS-CoV-2 RNA from the surfaces of different widely used clinical
and office supplies, decreasing their management risk and improving their reutilization.
Our findings support that ozone could provide an additional tool to control the spread of
the COVID-19 pandemic. The optimal treatment conditions (concentration and time of
ozone exposure) varied according to the composition and volume of the different materials.
Further research is required into the effects of the chronic exposure of these materials to
ozone, especially electronic components. The final real value of the procedure could also
depend on the variable costs and availability of the different materials to be treated. It is
necessary to develop new knowledge before extending the use of ozone in this context.
6. Patents
The initial ozonation chamber UVOZ
®
and a further update used in the study have
been patented (No. U202030703 and No. P202130273, respectively) by Lighting Dynamic
Technology, S.L., with the participation of 10 of the authors of this manuscript: F.R.A., B.C.,
E.C.-L., F.R.-E., J.E.P., J.V., A.B., J.L.M.-B., J.M.G.-M., P.S.-A., and J.L.-M.
Supplementary Materials:
The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/ijerph19148672/s1, Equation (S1): The destruction of ozone in the atmosphere.
Author Contributions:
Conceptualization, E.C.-L., F.R.-E., J.V., A.B., P.S.-A., J.L.-M. and B.C.; Formal
analysis, L.B.T.-M., E.C.-L., J.L.-M. and B.C.; Funding acquisition, J.L.-M. and B.C.; Investigation,
L.B.T.-M., E.C.-L., O.G.-P., F.R.-E., J.L.M.-B., J.M.G.-M., J.E.P., J.L.-M. and B.C.; Methodology, L.B.T.-M.,
E.C.-L., O.G.-P., F.R.-E., A.B., J.V., F.R.-A., J.E.P., J.L.-M. and B.C.; Project administration, J.L.-M. and
B.C.; Writing—original draft, L.B.T.-M., E.C.-L., A.B., J.L.-M. and B.C.; Writing—review and editing,
L.B.T.-M., E.C.-L., O.G.-P., F.R.-E., A.B., J.V., F.R.-A., J.L.M.-B., J.M.G.-M., P.S.-A., J.E.P., J.L.-M. and
B.C. All authors have read and agreed to the published version of the manuscript.
Funding:
The ozone therapy device Ozonobaric-P
®
(SEDECAL, Madrid, Spain) was funded by the
Instituto de Salud Carlos III, Madrid, Spain, and by the European Regional Development Funds
(FEDER) (#COV20/00702). The ozonation chamber UVOZ
®
was supported by Lighting Dynamic
Technology, S.L., Las Palmas, Spain. The acquisition of heat-inactivated SARS-CoV-2 strain, RT-PCR
measurements, and other disposables were funded by grants from: the Instituto de Salud Carlos
III, Madrid, Spain (#COV20/00702); Fundación Canaria del Instituto de Investigación Sanitaria de
Canarias (FIISC), Las Palmas, Spain (#PIFIISC20/19); Fundación Mapfre Guanarteme, Las Palmas,
Spain (#OA20/072); and Gobierno de Canarias, Las Palmas, Spain (#PI2020010357). During this work,
O.G.-P. was partially funded by the Instituto de Salud Carlos III, Madrid, Spain (#COV20/00702) and
by the Fundación Canaria del Instituto de Investigación Sanitaria de Canarias (FIISC), Las Palmas,
Spain (#PIFIISC20/19), and L.T-M. was partially funded by the Fundación Mapfre Guanarteme, Las
Palmas, Spain (#OA20/072) and Gobierno de Canarias, Las Palmas, Spain (#PI2020010357).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Int. J. Environ. Res. Public Health 2022,19, 8672 11 of 13
Acknowledgments:
J.V. was funded by CIBER—Consorcio Centro de Investigación Biomédica
en Red-de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain
(CB06/06/1088, PI19/00141), and by the European Regional Development’s Funds (FEDER). A.B.
collaborated thanks to RETOPROSOST-2-S2018/EMT-4459, funded by The Community of Madrid.
J.L.-M. was funded by RICET (RD16/0027/0001), CIBER CB21/13/00100 and Cabildo Insular de
Tenerife (Programa TF INNOVA 2016–2021), del Marco Estratégico de Desarrollo Insular (MEDI)
and FDCAN. The PPE components were contaminated using a heat-inactivated SARS-CoV-2 strain
(ATCCfi VR-1986HK
™
). This strain was deposited by the Centers for Disease Control and Prevention
and obtained through BEI Resources, NIAID, NIH: Genomic RNA from SARS-Related Coronavirus 2,
Isolate USA-WA1/2020, NR-52285.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
COVID-19 Coronavirus Disease 2019
CPAP Continuous positive airway pressure tube
DNA Deoxyribonucleic acid
O2Molecular oxygen
O3Ozone
PPE Personal protective equipment
RNA Ribonucleic acid
RT-qPCR Real-time polymerase chain reaction
SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
UV Ultraviolet
VERO African green monkey kidney epithelial cells
WHO World Health Organization
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