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Inactivation of Norovirus by ozone gas in conditions relevant to healthcare

Authors:

Abstract

We evaluated the ability of ozone gas to inactivate Norovirus and its animal surrogate feline calicivirus (FCV) in dried samples placed at various locations within a hotel room, a cruise liner cabin and an office. Norovirus was measured by quantitative reverse transcriptase real-time polymerase chain reaction (QRT-PCR) assay, and FCV by a combination of QRT-PCR and virus infectivity assays. We were able to reduce the concentration of infectious FCV by a factor of more than 10(3), and in some cases beyond detection, under optimal conditions of ozone exposure with less than an hour of total operation. QRT-PCR assays indicated similar decreases in both viral RNAs. Virus-containing samples dried onto hard surfaces (plastic, steel and glass), and soft surfaces such as fabric, cotton and carpet, were equally vulnerable to the treatment. Our results show that Norovirus can be inactivated by exposure to ozone gas from a portable commercial generator in settings such as hotel rooms, cruise ship cabins and healthcare facilities.
Inactivation of Norovirus by ozone gas
in conditions relevant to healthcare
J.B. Hudson
a,b,
*, M. Sharma
a
, M. Petric
b,c
a
Viroforce Systems Inc. Laboratory, Vancouver, Canada
b
Department of Pathology & Laboratory Medicine, University of British Columbia, Canada
c
British Columbia Centre for Disease Control, Vancouver, Canada
Received 21 September 2006; accepted 22 December 2006
KEYWORDS
Norovirus; Feline
calicivirus; Ozone;
Virucidal; Hotel; Cruise
liner; Healthcare;
QRT-PCR
Summary We evaluated the ability of ozone gas to inactivate Norovirus
and its animal surrogate feline calicivirus (FCV) in dried samples placed
at various locations within a hotel room, a cruise liner cabin and an office.
Norovirus was measured by quantitative reverse transcriptase real-time
polymerase chain reaction (QRT-PCR) assay, and FCV by a combination of
QRT-PCR and virus infectivity assays. We were able to reduce the concen-
tration of infectious FCV by a factor of more than 10
3
, and in some cases
beyond detection, under optimal conditions of ozone exposure with less
than an hour of total operation. QRT-PCR assays indicated similar
decreases in both viral RNAs. Virus-containing samples dried onto hard
surfaces (plastic, steel and glass), and soft surfaces such as fabric, cotton
and carpet, were equally vulnerable to the treatment. Our results show
that Norovirus can be inactivated by exposure to ozone gas from a portable
commercial generator in settings such as hotel rooms, cruise ship cabins
and healthcare facilities.
ª2007 The Hospital Infection Society. Published by Elsevier Ltd. All rights
reserved.
Introduction
Noroviruses (NVs, calicivirus family) are increas-
ingly recognized as the main cause of gastroenteritis
outbreaks in health care facilities, senior citizens’
homes and cruise ships. A challenge to developing
effective disinfectants against these environmen-
tally resistant agents has been the lack of a sensitive
methodology to quantify their infectivity. This has
been resolved in part by the use of the surrogate
feline calicivirus (FCV), which can be readily grown
and assayed in cell culture.
1
Moreover, it is now
feasible to correlate the virus titre with virus RNA
concentration as determined by quantitative re-
verse transcriptase real-time polymerase chain
reaction (QRT-PCR) assays.
2e6
Consequently, it is
possible to assess definitively the effect of antiviral
* Corresponding author. Address: UBC, Department of Pathol-
ogy and Lab Medicine, 2733 Heather Street, Vancouver, BC,
Canada V5Z 1M9. Tel./fax: þ1 604 267 0748.
E-mail address: jbhudson@interchange.ubc.ca
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doi:10.1016/j.jhin.2006.12.021
Journal of Hospital Infection (2007) -,1e6
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Please cite this article in press as: Hudson JB et al., Inactivation of Norovirus by ozone gas in conditions relevant to healthcare, J
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agents by comparing FCV and NV under similar inac-
tivation conditions.
2,4
In addition it is also feasible
to use FCV as a surrogate virus for NV in field condi-
tions where the experimental use of NV itself would
not be acceptable due to its infectious risk.
7
The use
of these techniques has allowed for significant
advances in our understanding of the epidemiology
and economic impact of NV infections.
8e11
NVs are very stable in the environment; hence,
agents such as bleach and peroxides are required
for disinfection, although the efficacy of such
agents against NV has been questioned.
2
More re-
cently it was shown that some of them were less
effective against calicivirus-contaminated fabrics
and carpet.
12
In addition, these agents are used
in liquid form, may not completely penetrate con-
taminated areas within a room and are labour-
intensive to use. Furthermore, they are difficult
to apply to surfaces such as bedding and curtains
where they may have a bleaching effect.
A recent study showed that ozone dissolved in
water could inactivate water-borne NV.
2
We there-
fore decided to evaluate the possibility of using
ozone gas as a virucidal agent against NV. This
was based on the premise that in view of the
superior penetrability of gases, it should be feasi-
ble to inactivate the virus in any location and on
any surface within a contaminated room. We
therefore tested the efficacy of ozone gas, from
a proprietary portable ozone generator, against
FCV- and NV-containing specimens, in an office,
a hotel room and a cruise liner cabin. We expected
that the findings from such tests would be
relevant to any healthcare facility, which
could be temporarily isolated for treatment with
ozone gas.
Methods
Equipment
The prototype Viroforce ozone generator was
constructed as a portable module containing mul-
tiple corona discharge units, a circulating fan and
an efficient catalytic converter (scrubber) to re-
convert ozone to oxygen at the termination of the
ozone exposure period. The unit was controlled
remotely from outside the test room. In addition,
a portable custom-made rapid humidifying device
(RHD) was used to provide a burst of water vapour
when required. Preliminary tests in our laboratory
had indicated that an ozone level of 25 ppm, at
high relative humidity (in excess of 70%), resulted
in more than 99.9% inactivation of several viruses,
including feline calicivirus (unpublished data).
Ozone concentration was monitored continu-
ously by means of an Advanced Pollution Instru-
mentation Inc. model 450 system (from Teledyne,
San Diego, CA, USA), which measured samples of
the ozonated air and passed them through a UV
spectrometer. The input teflon sampling tube was
taped in an appropriate location for the duration
of the experiment.
Relative humidity and temperature were re-
corded by a portable hygrometer (VWR Scientific,
Ontario, Canada). The probe was taped in a con-
venient location inside the test room. Tempera-
ture did not exceed 23 C.
Test rooms and protocol
Most tests were carried out in an office (volume
34 m
3
) containing normal office furniture and adja-
cent to the laboratory. Additional tests were con-
ducted in a local standard hotel room (volume
47.6 m
3
) containing double bed, table, chairs,
open closet and adjoining bathroom, and in a stan-
dard cabin (volume 36.4 m
3
) of a cruise liner which
was docked in Vancouver for half a day. Vents,
windows, and doors were sealed with tape.
The standard protocol was based upon prelim-
inary laboratory tests: virus samples (50e100 mL)
were dried onto sterile plastic or other surfaces
in duplicate in the Viroforce Laboratory. When
dry, the samples were transported quickly to the
test site in sterile containers. The samples were
placed at various locations in the test room, and
the ozone generator and rapid humidifying device
(RHD) were placed in a central location. These
units were operated remotely from outside the
room. At the commencement of the test, the sam-
ples were uncovered, the door closed and sealed,
and the generator switched on.
The ozone level reached 20e25 ppm within sev-
eral minutes and was maintained at this level for
20 min. This time was determined from consultation
with cruise liner staff, who indicated the desirabil-
ity of a short treatment time compatible with the
rapid turnover of passengers. The RHD was then ac-
tivated to produce a burst of water vapour for 5 min.
Both generator and RHD were then switched off for
another 10 min to allow ‘incubation’ in the humid
atmosphere. The scrubber was then turned on to re-
move all ozone gas. Ozone levels decreased to less
than 1 ppm within 15 min, at which point the door
was opened and the test samples retrieved for
transport back to the laboratory. The samples
were reconstituted in 1.0 mL medium and kept
frozen at e80 C until ready for assay.
In all experiments, control samples of the dried
viruses were either transported to the test site, and
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stored away from the ozone, or were stored in the
Viroforce laboratory for the duration of the tests.
Materials
The lids of sterile polystyrene tissue culture trays
were used as plastic surfaces. Samples of fabrics
and carpet (typical of those used in hotel rooms)
were cut into small pieces, cleaned in detergent,
washed, dried, and sterilized by autoclaving. Cot-
ton tips were heated for 2 min in a microwave oven.
All media, serum and other reagents used in
cell and virus work were obtained from Invitrogen
(Gibco, Ontario, Canada). Sterile plastic culture
vessels and other supplies were BD-Falcon brand
obtained from VWR Scientific. Molecular biology
reagents and kits were obtained from Qiagen
(Missisauga, Ontario, Canada) and PCR primers
from Operon (Huntsville, AL, USA).
Cell lines and viruses
FCV and feline kidney cell line (FK cells) were
obtained from BC-Centre for Disease Control
(BCCDC). The cells were grown in Dulbecco’s
modified Eagle’s medium with 5% fetal bovine
serum, without antibiotics or antimycotics. Stock
virus, obtained as clarified supernatants from FCV-
infected flasks, had titres of 1e210
7
Pfu (plaque
forming units)/mL.
NV specimens, in the form of stools in which NV
had been diagnosed by RT-PCR, were obtained from
BC-CDC, along with NV-negative stool samples.
Virus assays
For the quantitative measurement of infectious
FCV, plaque assays were conducted in duplicate,
according to standard techniques making use of
agarose overlays.
1
Very low numbers of infectious
virus were detected by incubating undiluted sam-
ples and serial two-fold dilutions along rows of cells
cultured in 96-well trays (without overlays). The
end-point was the dilution of sample that no longer
gave rise to characteristic viral CPE (cytopathic ef-
fects). If no viral CPE were seen in any culture wells
after 5 days of incubation, the sample was consid-
ered completely free of infectious virus.
13
Quantitative RT-PCR (reverse
transcriptase real-time PCR)
measurements
Treated and control samples were removed from
frozen storage and their RNA extracted and
purified by means of Qiagen RNA extraction kits.
Quantitative RT-PCR measurements were made on
the Opticon DNA engine. Methods followed those
described in Frankhauser et al. and Scansen
et al.
6,8
Primers for FCV were: forward primer 5’-TGGA
TGAACTACCCGCCA; reverse primer 5’-GCACATCAT
ATGCGGCTC; and for NV strains the following pairs
were used):
MON 431 tgg acI agR ggI ccY aaY ca, RNA sense;
MON 432 tgg acI cgY ggI ccY aaY ca, RNA sense;
MON 433 gaa Yct cat cca Yct gaa cat, DNA sense;
MON 434 gaa Scg cat cca Rcg gaa cat, DNA sense;
where I ¼Inosine; R ¼puRine (A/G); Y ¼
pYrimidine(C/T); S ¼Strong (C/G).
Results
Office tests
Replicate samples of FCV and NV (three different
stool samples) were tested in the office. In some
samples of FCV, fetal bovine serum or NV-positive
stool was added (1:1) to determine the effect of
a representative organic load on the treatment.
Following the standard ozone protocol, samples
were assayed for infectious FCV and for viral RNA
by QRT-PCR (Table I).
Substantial inactivation of FCV and NV samples
was achieved, with a comparable reduction in
RT-PCR values, indicating that infectivity of both
viruses would be similarly affected if it were
possible to assay for NV infectivity. This was an
important finding since it would be necessary to
conduct subsequent tests in hotel rooms and
cabins with FCV only for practical reasons.
Table I Field test in office
Virus Pfu
(fraction
of control)
Log
10
RT-PCR
(fraction
of control)
Log
10
FCV 0.012 1.92 0.029 1.54
FCV, þFBS (1:1) 0.017 1.77 0.021 1.68
FCV, þ
stool (1:1)
0.015 1.82 0.020 1.70
NV sample 1 _ 0.070 1.15
NV sample 2 _ 0.055 1.26
NV sample 3 _ 0.046 1.34
Control values: for FCV infectivity, 5.1 10
4
Pfu/mL; and for
PCR, 116 to 218 ng RNA; for NV samples, NV 1 ¼58.15 ng
RNA, NV 2 ¼129.5 ng RNA, NV 3 ¼114.1 ng RNA.
Pfu, plaque forming units; FCV, feline calicivirus (FCV); RT-
PCR, reverse transcriptase real-time polymerase chain
reaction; Norovirus (NV).
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In addition the presence of added serum and
stool (from an NV-positive sample) did not
adversely affect the response of FCV to ozone
treatment (Table I). Figure 1 illustrates the gel
electrophoresis patterns of amplified RNA
from the three NV samples. The bands from all
the ozone-treated samples were substantially
reduced in intensity, indicating that the treated
virus particles had been disrupted, although
some residual pieces of viral RNA were still pres-
ent and capable of being amplified in the PCR
reactions.
Hotel room
Replicate samples of FCV were placed in three
different locations, one in the bathroom next to
the sink, one on top of the bed and the third on top
of the table.
Samples were subsequently assayed for virus
infectivity and QRT-PCR. The results are shown in
Table II. The FCV samples that gave no virus pla-
ques at 1:10 dilution (bathroom and table samples)
were reassayed by the CPE end-point dilution test
to determine if there were any infectious viruses
remaining after treatment. Control and bed sam-
ples were also assayed for comparison. Since the
bathroom and table samples gave no CPE in these
assays, we concluded that for these samples the
virus had been eradicated.
Cruise liner cabin
Two tests were carried out with FCV on plastic
trays. The first test used the standard protocol,
and the second one used a more compact schedule
that would be more attractive to cruise liner
schedules.
Samples were placed on top of the bed, on the
table and in the adjoining bathroom. Results from
both tests were essentially the same, indicating
that the abbreviated protocol could work with the
desired efficiency. Data from test no. 2, the
abbreviated schedule, are shown in Table III.In
this test, the operation times were reduced to
15 min ozone, followed by 4 min water vapour
pulse (RHD), no post-humidity ‘incubation’ period,
and 15 min scrubber.
No residual infectious virus was detected in the
CPE tests. Thus the virus had been eradicated in
these samples (inactivation by a factor of more
than 10
4
). The corresponding RT-PCR measure-
ments showed residual amplified viral RNA at
a level of 0.03 or less (Table III).
Virus on soft surfaces
For additional tests conducted in the office,
replicate samples of both FCV- and NV-positive
stools were dried onto plastic trays as usual and
also onto samples of fabric, cotton and carpet.
These were placed at various locations within the
office to mimic possible contamination sites during
an NV outbreak. The standard ozone exposure
protocol was used and subsequently samples
were assayed for FCV infectivity and for viral
RNA by QRT-PCR. The results are summarized in
Table IV.
(A)
12345678910
(B)
12345678
Figure 1 Electrophoresis of PCR-amplified Norovirus
(NV) RNA (213 base pair band). (A) Lane 5, NV-1, no ozone;
lane 6, NV-1, ozone-treated; lane 7, NV-2, no ozone; lane
8, NV-2, ozone-treated. Other lanes: 1, DNA base pair lad-
der; 2e4, NV-negative stool samples; 9, 10, NV-negative
samples. (B) Lane 1, DNA base pair ladder; lane 2, NV-3,
no ozone; lane 2, lane 3, NV-3, ozone-treated. Other
lanes: 4e7, NV-negative stool samples; 8, NV-positive
stool sample.
Table II Field test in hotel room
Virus Pfu (fraction of control) Viral CPE at dilution Log
10
RT-PCR (fraction of control) Log
10
FCV, bathroom 0 None <4.0 0.077 1.11
FCV, bed <0.0002 1:8 <3.7 0.077 1.11
FCV, table 0 None <4.0 0.075 1.12
Control values 8.0 10
4
Pfu/mL >1:4096 415.5 ng
Pfu, plaque forming units; CPE, cytopathic effects; RT-PCR, reverse transcriptase real-time polymerase chain reaction; FCV,
feline calcivirus.
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All samples showed similar sensitivity to ozone,
regardless of their location or the surface onto
which they were dried.
Discussion
We have shown that we can inactivate Norovirus
contained in dried stool samples in an office,
a hotel room and a cruise liner cabin. In compar-
ison with the feline calicivirus, a generally accept-
able surrogate virus for the evaluation of NV titres,
it is reasonable to conclude from our data that
we were able to achieve inactivation of virus by
a factor of more than 10
3
, and, under optimal con-
ditions, to eradicate the virus.
The QRT-PCR technique, currently the only
convenient method for measuring NV, does not
measure virus infectivity per se, but rather a de-
fined sequence of the viral genome, which one
would expect to be more resistant to the damaging
effects of ozone gas than infectivity. Consequently,
this nucleic-acid-based technique probably under-
estimates the effectiveness of antiviral agents,
hence the need for comparison with a related virus
that can be assayed for infectivity as well as by
QRT-PCR. The finding that our treatment protocol
could in many cases eradicate FCV infectivity
indicates that under the same conditions NV should
also be rendered non-infectious, even though its
genome may still be partly intact.
Addition of serum and NV-positive stool to FCV
samples did not adversely affect their sensitivity to
ozone, an observation that confirmed the validity
of FCV as a surrogate for NV in locations where it
would be impractical to use live NV samples.
Virus samples dried onto soft furnishings were
also vulnerable to ozone. The degree of virus
inactivation was comparable to that observed for
samples on plastic, confirming that any virus de-
posited onto curtains, bedding, linen and chair
covers, etc. should not present a barrier to in-
activation. Recently, it was reported that some
liquid disinfectants were not very effective against
FCV on fabrics and carpet.
12
We had previously
shown in laboratory tests that virus dried onto
other hard surfaces such as glass and steel could
be inactivated by ozone gas as readily as on plastic
(unpublished results).
Ozone gas has several advantages as a practical
antiviral agent. It can effectively penetrate every
part of a room, including sites that might prove
difficult to gain access to with conventional liquids
and manual cleaning procedures. For example, in
our tests, virus deposited under the table or
adsorbed to fabric taped to a window were just
as vulnerable to attack as virus placed in more
accessible sites. The gas is easy and economical to
produce, and is a natural compound which decays
quickly back to oxygen with a half-life of about
20 min. The use of a catalytic converter (scrubber)
considerably speeds up the removal of the gas. In
addition, in the event of possible malfunction dur-
ing application, the gas is readily detected by
smell and hence can be avoided.
Its major disadvantage is its potential toxicity
at high concentration, which precludes its use in
areas continuously populated by people. In prac-
tice this means it can only be used in rooms that
can be sealed off or quarantined for the duration
of the treatment. Since the standard protocol
requires less than an hour to perform, however,
Table III Cruise liner cabin. Feline calicivirus (FCV) infectivity
Sample Infectivity (Pfu/mL) Surviving fraction RT-PCR surviving fraction
Control 5.37 10
4
1.0 1.0
Treated (bathroom, bed and table) <10
1
<0.0002 0.003e0.03
Pfu, plaque forming units; RT-PCR, reverse transcriptase real-time polymerase chain reaction.
Table IV Norovirus (NV) and surrogate feline calicivirus (FCV) on different surfaces
Sample type FCV infectivity
fraction of control
FCV QRT-PCR
fraction of control
NV QRT-PCR
fraction of control
Plastic: table top, underside of
table and wall
All 610
5
0.0013e0.0016 0.05e0.069
Fabric: table top, wall, window All 310
4
0.0036e0.0048 0.056e0.065
Cotton: table top, different locations All 310
5
0.076e0.079 0.030e0.031
Carpet: floor, different locations All 410
5
0.0028e0.0032 0.042e0.059
Control values 2.7e3.6 10
5
Pfu 18.7e57.3 ng RNA 98.6e132.7 ng RNA
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this should not be a barrier to utilization given its
potential efficacy.
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... 33 The efficacy of ozone gas in an office, a laboratory, a hotel room and a cruise liner cabin was also tested. 7,25 Overall, the variation in ozonation protocols among the studies did not allow direct comparison of results. ...
... 7,20-24,28,31,32,33 Among them, eight evaluated the influence of RH on virus degradation and/or virus infectivity. 7,21,24,25,28,29,31,32 The results of these experiments controlling RH demonstrated that RH plays a key role in ozone reactivity, and in most studies, the optimum efficacy of ozone treatment was achieved under high RH (> 50%). All of these studies reported a positive effect of elevated RH on the overall efficacy of ozone treatment. ...
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Abstract The aim of this scoping review was to provide sufficient information about the effectiveness of ozone gas in virus inactivation of surfaces and objects under different environmental conditions. The review was performed according to the list of PRISMA SrC recommendations and the JBI Manual for Evidence Synthesis for Scoping Reviews. The review was registered in Open Science Framework (OSF). EMBASE (Ovid), Lilacs, LIVIVO, MEDLINE (PubMed), SciELO, Scopus and Web of Science were primary sources, and “gray literature” was searched in OpenGray and OpenThesis. A study was included if it reported primary data on the effect of ozone gas application for vehicle-borne and airborne virus inactivation. No language or publication date restriction was applied. The search was conduct on July 1, 2020. A total of 16,120 studies were screened, and after exclusion of noneligible studies, fifteen studies fulfilled all selection criteria. Application of ozone gas varied in terms of concentration, ozone exposure period and the devices used to generate ozone gas. Twelve studies showed positive results for inactivation of different virus types, including bacteriophages, SARS-CoV-2 surrogates and other vehicle-borne viruses. Most of the studies were classified as unclear regarding sponsorship status. Although most of the population has not yet been vaccinated against COVID-19, disinfection of environments, surfaces, and objects is an essential prevention strategy to control the spread of this disease. The results of this Scoping Review demonstrate that ozone gas is promising for viral disinfection of surfaces.
... Some traditional disinfecting hospital environments' apparent lack of effectiveness [47,48] stimulated the search for new decontamination methods that are also "environmentally friendly." As a result, there has been engagement in using ozone gas as a chemical element for antimicrobial control in several areas [49][50][51][52][53][54] and as a disinfectant [55][56][57][58]. ...
... Ozone generated by ozone purifiers in low concentrations is safe when following the manufacturer's scheme [71]. Gaseous ozone in relatively high concentrations (25 parts per million-ppm) has also been used to inactivate norovirus and bacteria in office and hotel rooms, removing this ozone after using a system purifier [51,52]. In medicine, O 3 has already been used in different forms of application (parenteral or local), aiming to combat ischemia, joint diseases, immunosuppression, degenerative diseases, and infections [72][73][74], and for therapeutic purposes [75,76]. ...
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(1) Background: Disinfection of medical devices designed for clinical use associated or not with the growing area of tissue engineering is an urgent need. However, traditional disinfection methods are not always suitable for some biomaterials, especially those sensitive to chemical, thermal, or radiation. Therefore, the objective of this study was to evaluate the minimal concentration of ozone gas (O3) necessary to control and kill a set of sensitive or multi-resistant Gram-positive and Gram-negative bacteria. The cell viability, membrane permeability, and the levels of reactive intracellular oxygen (ROS) species were also investigated; (2) Material and Methods: Four standard strains and a clinical MDR strain were exposed to low doses of ozone at different concentrations and times. Bacterial inactivation (cultivability, membrane damage) was investigated using colony counts, resazurin as a metabolic indicator, and propidium iodide (PI). A fluorescent probe (H2DCFDA) was used for the ROS analyses; (3) Results: No reduction in the count colony was detected after O3 exposure compared to the control group. However, the cell viability of E. coli (30%), P. aeruginosa (25%), and A. baumannii (15%) was reduced considerably. The bacterial membrane of all strains was not affected by O3 but presented a significant increase of ROS in E. coli (90 ± 14%), P. aeruginosa (62.5 ± 19%), and A. baumanni (52.6 ± 5%); (4) Conclusion: Low doses of ozone were able to interfere in the cell viability of most strains studied, and although it does not cause damage to the bacterial membrane, increased levels of reactive ROS are responsible for causing a detrimental effect in the lipids, proteins, and DNA metabolism.
... In intensive care units (ICUs), even after adopting strict cleaning and disinfection protocols, many patients are infected with HAIs [6][7][8]. These infections are more frequent in these units, where outbreaks usually originate [9] since the use of antibiotics is a chemical element for antimicrobial control in several areas [48][49][50][51][52][53] and as a disinfectant [54][55][56][57]. ...
... However, O3 has already been applied to clean hospital clothing [59], and a recent study demonstrated the eradication of methicillin-resistant Staphylococcus aureus (MRSA) in a nurse's home environment after O3 decontamination [49]. Gaseous ozone in relatively high concentrations (25 parts per million -ppm) has also been used to inactivate norovirus and bacteria in office and hotel rooms, removing this ozone after using a system purifier [50,51]. In medicine, O3 has already been used in different forms of application (parenteral or local), aiming to combat ischemia, joint diseases, immunosuppression, degenerative diseases, and infections [60][61][62] and for therapeutic purposes [63,64]. ...
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(1) Background: Disinfection of medical devices designed for clinical use associated or not with the growing area of tissue engineering is an urgent need. However, traditional disinfection methods are not always suitable for some biomaterials, especially those sensitive to chemical, thermal, or radiation. Therefore, the objective of this study was to evaluate the minimal concentration of ozone gas (O3) necessary to control and kill a set of sensitive or multi-resistant Gram-positive and Gram-negative bacteria. The cell viability, membrane permeability, and the levels of reactive intracellular oxygen (ROS) species were also investigated; (2) Material and Methods: Four standard strains and a clinical MDR strain were exposed to low doses of ozone at different concentrations and times. Bacterial inactivation (cultivability, membrane damage) was investigated using colony counts, resazurin as a metabolic indicator, and propidium iodide (PI). A fluorescent probe (H2DCFDA) was used for the ROS analyses; (3) Results: No reduction in the count colony was detected after O3 exposure, compared to the control group. However, the cell viability of E. coli (30%), P. aeruginosa (25%), and A. baumannii (15%) was reduced considerably. The bacterial membrane of all strains was not affected by O3 but presented a significant increase of ROS in E. coli (90 ± 14%), P. aeruginosa (62.5 ± 19%), and A. baumanni (52.6 ± 5%); (4) Conclusion: Low doses of ozone were able to interfere in the cell viability of most strains studied, and although it does not cause damage to the bacterial membrane, increased levels of reactive ROS are responsible for causing a detrimental effect in the lipids, proteins, and DNA metabolism.
... •-, and singlet oxygen, potentially increasing virus inactivation (Dubuis et al., 2020;Hudson et al., 2007). It can thus be concluded that humidity has a strong effect on disinfection of dried virus samples and its combination with ozone yields increased virus inactivation (Hudson et al., 2009;Elford and van den Ende, 1942). ...
Article
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This study evaluated the inactivation of SARS-CoV-2, the virus responsible for COVID-19, by ozone using virus grown in cell culture media either dried on surfaces (plastic, glass, stainless steel, copper, and coupons of ambulance seat and floor) or suspended in liquid. Treatment in liquid reduced SARS-CoV-2 at a rate of 0.92±0.11 log10-reduction per ozone CT dose(mg.min/L); where CT is ozone concentration times exposure time. On surface, the synergistic effect of CT and relative humidity (RH) was key to virus inactivation; the rate varied from 0.01 to 0.27 log10-reduction per ozone CT value(g.min/m³) as RH varied from 17% to 70%. Depletion of ozone by competitive reactions with the medium constituents, mass transfer limiting the penetration of ozone to the bulk of the medium, and occlusion of the virus in dried matrix were postulated as potential mechanisms that reduce ozone efficacy. RH70% was found plausible since it provided the highest disinfection rate while being below the critical RH that promotes mould growth in buildings. In conclusion, through careful choice of (CT, RH), gaseous ozone is effective against SARS-CoV-2 and our results are of significance to a growing field where ozone is applied to control the spread of COVID-19.
... Thus, ozone is a natural compound, easily produced in place from oxygen or air, and is divided into oxygen for a half-life of approximately 20 minutes (± 10 minutes depending on the environment). As a gas, it can penetrate much more efficiently than manually applied liquid sprays and aerosols in all areas in a room, including crevices, fixtures, fabrics, and furniture substrates [32][33][34]. The only major drawback is its wear and potential toxicity to humans when exposed for a long time to certain materials, such as natural rubber. ...
Article
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Tuberculosis and in some cases, flu, colds and other airborne diseases. Since research is one of the biggest concerns of causing influenza pandemics, most research surrounding aerosol contamination revolves around environmental influences on the influenza virus. Many literatures suggest that influenza is transmitted primarily through close contact, such as exposure to large respiratory droplets, direct mouth-to-mouth contact and short-term exposure to infectious aerosols. Diffusion can be accelerated or controlled by heating, ventilation and air conditioning (HVAC) systems. Researches continue that advances state of knowledge in the specific techniques that control airborne infectious disease transmission through HVAC systems, including ventilation rates, airflow regimes, filtration, and ultraviolet germicidal irradiation (UVGI). In this paper three methods of transmission of Airborne Infectious Diseases are discussed, namely through direct contact, large droplet contact, inhalation of droplet core. An extensive literature review of many papers was conducted infectious diseases spread in several different ways and the transmission of infectious viruses. This review targets direct and indirect contact as well as infectious viruses known to be transmitted from the air. And he focused on preventive ventilation systems for these targets. This paper will give idea to support further research on engineering controls to reduce infectious disease transmission.
... Between subjects, the chamber was sterilised using an ozone generator, which has been shown to effectively remove both viruses [24] and bacteria [25]. The mask, tube and plug were thoroughly washed with water and soap and then disinfected. ...
Article
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Respiratory aerosols from breathing and talking are an important transmission route for viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous studies have found that particles with diameters ranging from 10 nm to 145 μm are produced from different regions in the respiratory system and especially smaller particles can remain airborne for long periods while carrying viral RNA. We present the first study in which respiratory aerosols have been simultaneously measured with carbon dioxide (CO2) to establish the correlation between the two concentrations. CO2 concentrations are easily available through low-cost sensors and could be used to estimate viral exposure through this correlation, whereas source-specific aerosol measurements are complicated and not possible with low-cost sensors. The increase in both respiratory aerosols and CO2 was linear over ten minutes in a 2 m3 chamber for all participants, suggesting a strong correlation. On average, talking released more particles than breathing, with 14,600 ± 16,800 min−1 (one-σ standard deviation) and 6210 ± 5630 min−1 on average, respectively, while CO2 increased with 139 ± 33 ppm min−1 during talking and 143 ± 29 ppm min−1 during breathing. Assuming a typical viral load of 7×106 RNA copies per mL of oral fluid, ten minutes of talking and breathing are estimated to produce 1 and 16 suspended RNA copies, respectively, correlating to a CO2 concentration of around 1800 ppm in a 2 m3 chamber. However, viral loads can vary by several orders of magnitude depending on the stage of the disease and the individual. It was therefore concluded that, by measuring CO2 concentrations, only the number and volume concentrations of released particles can be estimated with reasonable certainty, while the number of suspended RNA copies cannot.
... The inhibitory and lethal effects of ozone on pathogenic microorganisms have been observed since the 19th century, and the most cited explanation of these effects is based on the disruption of their envelopes through peroxidation of phospholipids and the interaction with proteins [31]. As mentioned by H u d s o n et al. [8], the gas state allows ozone to get to areas that are difficult to reach and to disinfect much more than just surfaces. ...
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The aim of this study was to observe the efficiency of ozone transferred by an airstone bubble diffuser, using two ozone generators with different output of ozone (5 g.h–1 ‒ G1; 15 g.h–1 ‒ G2). The retention of ozone in ozonised distilled and potable water and the devitalisation effects on E. coli in the water were also noted. Ozone was introduced to two types of potable water of different composition intended for mass consumption, (MC)a and (MC)b, distilled water, and well water intended for individual consumption. The devitalisation effect of ozone on E. coli in well water (WW) and added to potable and distilled water was observed. The results of our study showed that under the conditions used, the level of ozone during ozonisation with G1 increased more rapidly in distilled water and after termination of ozonisation, the retention of ozone in distilled water was a little lower in comparison with the potable water. The devitalisation of E. coli either naturally present in the water or added to it required the level of ozone close to or equal to 0.25 mg.l–1.
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Ozone has been used for surface disinfection to contain bacterial, fungal, mold, and certain viral infections; however, the use of ozone generated from nonthermal plasma devices have not been thoroughly investigated for surface disinfection. Here, we aimed to determine the impact of nonthermal plasma‐generated ozone (PGO) on the coronavirus. Human coronavirus 229E was exposed to PGO and its infectivity was evaluated. PGO exposure of approximately 7 ppm reduced the viral titer after 4 h. Our results indicate that PGO exposure not only reduces the expression of the viral nucleocapsid gene and spike glycoprotein levels but may also stimulate the expression of the antiviral response gene in host cells. These findings can thus be useful to support existing surface disinfection methods. HCoV‐229E virus, a surrogate of SARS‐CoV‐2, was exposed to cold plasma‐generated ozone for 4 h under ambient room conditions. A significant reduction of viral infectivity in host MRC‐5 cells was observed. Exposure to plasma‐generated ozone led to a reduction in the expression of viral nucleocapsid genes and a spike in glycoprotein levels. Also, enhanced expression of antiviral response genes was observed in host cells.
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This paper reports a plasma reactive oxygen species (ROS) method for decontamination of PPE (N95 respirators and gowns) using a surface DBD source to meet the increased need of PPE due to the COVID-19 pandemic. A system is presented consisting of a mobile trailer (35 m3) along with several Dielectric barrier discharge sources installed for generating a plasma ROS level to achieve viral decontamination. The plasma ROS treated respirators were evaluated at the CDC NPPTL, and additional PPE specimens and material functionality testing were performed at Texas A&M. The effects of decontamination on the performance of respirators were tested using a modified version of the NIOSH Standard Test Procedure TEB-APR-STP-0059 to determine particulate filtration efficiency. The treated Prestige Ameritech and BYD brand N95 respirators show filtration efficiencies greater than 95% and maintain their integrity. The overall mechanical and functionality tests for plasma ROS treated PPE show no significant variations.
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The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100 degrees C. UV-B radiation from 0 to 150 mJ/cm(2) caused dose-dependent inactivation, with a 3 D (D = 1 log(10)) reduction in infectivity at 34 mJ/cm(2) for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.
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Healthcare-associated outbreaks of gastroenteritis are an increasingly recognized problem, but detailed knowledge of the epidemiology of these events is lacking. We actively monitored three hospital systems in England for outbreaks of gastroenteritis in 2002 to 2003. A total of 2,154 patients (2.21 cases/1,000-hospital-days) and 1,360 healthcare staff (0.47 cases/1,000-hospital-days) were affected in 227 unit outbreaks (1.33 outbreaks/unit-year). Norovirus, detected in 63% of outbreaks, was the predominant etiologic agent. Restricting new admissions to affected units resulted in 5,443 lost bed-days. The cost of bed-days lost plus staff absence was calculated to be 635,000 pounds sterling (US. 1.01 million dollars) per 1,000 beds. By our extrapolation, gastroenteritis outbreaks likely cost the English National Health Service 115 million pounds sterling (US 184 million dollars) in 2002 to 2003. Outbreaks were contained faster (7.9 vs. 15.4 days, p = 0.0023) when units were rapidly closed to new admissions (<4 days). Implementing control measures rapidly may be effective in controlling outbreaks.
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Feline calicivirus (FCV) has been used by researchers as a surrogate for Norwalk virus (NV), since they share a similar genomic organization, physicochemical characteristics, and are grouped in the same family, Caliciviridae. Unlike NV, however, FCV can grow in established cell lines and produce a syncytial form of cytopathic effect. In this report, we describe the development and standardization of a plaque assay for FCV using monolayers of an established line of feline kidney (CrFK) cells in 12-well cell culture plates. The assay method has demonstrated reproducibility, ease of performance and resulted in clear plaque zones, readable in 24 h after virus inoculation. The infectivity titre of the virus by this plaque assay agreed well with tissue culture infectious dose(50) (TCID(50)) determinations. The described plaque assay would be a valuable tool in conducting various quantitative investigations using FCV as a model for NV and Norwalk-like viruses (NLV).
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This report describes a feline calicivirus (FCV) p30 gene-based real-time SYBR Green I reverse transcriptase polymerase chain reaction (RT-PCR) assay that is capable of detecting low virus concentrations and a broad range of FCV isolates. The assay consisted of a 1-step RT-PCR reaction with primers delineating a 126-base-pair (bp) region of the FCV p30 gene. Sensitivity of the RT-PCR assay was determined to be equivalent to a FCV titer of 1.2 x 10(1) to 1.2 x 10(2) TCID50/mL. The assay was linear over a wide range of template concentrations and had a reaction efficiency of 95%. Specific FCV amplification products were detected from 51 wild-type FCV isolates, whereas specific products were not detected from a canine calicivirus, a rabbit calicivirus, and a bovine calicivirus. The primers used in this study amplified a large number of North American FCV isolates and further confirm the diagnostic utility of p30 gene-based real-time RT-PCR for detection of FCV.
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A reverse transcriptase polymerase chain reaction assay was used to study the transfer of Norovirus (NV) from contaminated faecal material via fingers and cloths to other hand-contact surfaces. The results showed that, where fingers come into contact with virus-contaminated material, NV is consistently transferred via the fingers to melamine surfaces and from there to other typical hand-contact surfaces, such as taps, door handles and telephone receivers. It was found that contaminated fingers could sequentially transfer virus to up to seven clean surfaces. The effectiveness of detergent- and disinfectant-based cleaning regimes typical of those that might be used to decontaminate faecally contaminated surfaces and reduce spread of NV was also compared. It was found that detergent-based cleaning with a cloth to produce a visibly clean surface consistently failed to eliminate NV contamination. Where there was faecal soiling, although a combined hypochlorite/detergent formulation at 5000 ppm of available chlorine produced a significant risk reduction, NV contamination could still be detected on up to 28% of surfaces. In order consistently to achieve good hygiene, it was necessary to wipe the surface clean using a cloth soaked in detergent before applying the combined hypochlorite/detergent. When detergent cleaning alone or combined hypochlorite/detergent treatment failed to eliminate NV contamination from the surface and the cleaning cloth was then used to wipe another surface, the virus was transferred to that surface and to the hands of the person handling the cloth. In contrast, were surfaces where contaminated with NV-infected faecal suspension diluted to 1 in 10 and 1 in 80, intended to simulate surfaces that have become contaminated after secondary transfer, treatment with a combined bleach/detergent formulation, without prior cleaning, was sufficient to decontaminate surfaces and prevent transfer.
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To compare 5 methods of preparation of RNA from feline urine samples for use in a feline calicivirus (FCV), p30 gene-based, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay. Urine and blood samples from 6 specific-pathogen-free cats. Aliquots of each urine sample (unmodified, centrifuged, or mixed with whole or hemolyzed blood) were spiked with FCV and serially diluted in urine. Serial dilutions of FCV in tissue culture medium were used as positive controls. Viral RNA was prepared via dilution and thermal inactivation (DT method), polyethylene glycol precipitation (PEG method), isolation with oligo(dT)25-coated magnetic beads (dTMB method), or extraction by use of 2 silica gel-based columns (RN or QA method). Lower detection limits and mean RT-PCR threshold cycle (Ct) values associated with each RNA preparation method and sample type were compared. Because DT-prepared samples yielded negative results via RT-PCR assay, this method was not evaluated. Lower detection limits (TCID50/sample) for the assay in urine were 1950, 104, 11, and 7 for PEG-, dTMB-, RN-, and QA-prepared samples, respectively. For RN and QA preparations, Ct values were similar and significantly lower than those for dTMB and PEG preparations. Overall, urine modifications did not affect FCV RNA detection in dTMB-, QA-, and RN-prepared samples. Of the methods evaluated, the RN and QA methods of RNA preparation were most appropriate for the FCV RT-PCR assay. An RT-PCR assay optimized for detection of FCV in feline urine may aid investigations of FCV-induced urinary tract diseases in cats.