Keywords
ozone - ambiental sanitization - airborne particles - HEPA filter - clean rooms
Introduction
The COVID-19 pandemic is a great concern and threat to the population worldwide and
a huge challenge for healthcare professionals. In such times of major health crises,
special attention is given to the importance of proper sanitization control of common
environments, considering that air can be a vehicle for infectious agents.[1] Decontamination of the air of indoor health facilities is crucial in maintaining
public health and requires continuous surveillance, improvement, and expansion of
access.[2]
[3]
Monitoring the air quality of indoor health facilities and removing airborne particles
from such environments is recommended.[2]
[3] Control of environmental air can be performed by counting viable and non-viable
particles. Non-viable particles are those that are unable to survive, grow, develop,
and reproduce. Although airborne microorganisms are not free-floating or single cells,
they often associate with 10 to 20 µm particles.[4]
Particles classified as viable or non-viable can come from different sources, such
as flaking of the skin in humans or due to movement when opening packages, for instance.[4] The method of counting viable particles has always been used to control the sterility
of environmental air, as seen, for example in previous research.[5] They analyzed microbial contamination of the air in operating rooms of a Ghanaian
hospital with an active air MAS-100 sampler. The authors verified the count of viable
particles in 124 elective surgical procedures, both clean and contaminated. The study
revealed an increased number of viable airborne particles during the procedures, exceeding
the levels established by the current health legislation.[5]
This methodology, although functional, has some disadvantages, such as cost, a longer
time for reading the results, and biological risk of the operators who manipulate
live microorganisms. With the emergence of modern portable particle counters, the
daily count of non-viable particles is now being used in a variety of critical areas
because it is simple and inexpensive. The implementation of this monitoring procedure
allows for a real-time response to any deviation in the number of dispersed particles
in the environment.[6]
For these reasons, counting non-viable particles is a reliable diagnostic tool for
measuring air quality. Furthermore, it presents a lower biological risk, is reproducible,
and is cheaper than microbial counting. The main goal of monitoring non-viable particles
is to demonstrate that a cleanroom is operating within the pre-established limits
without directly evaluating the environment's sterility. It can, therefore, be used
as a predictive parameter in monitoring contamination. The relationship of non-viable
particles and viable airborne particles, concluding that both are indeed correlated,
opens the possibility to use the non-viable particle count as an effective method
of environmental monitoring.[6]
Therefore, there is a tendency to apply the standards for non-viable particle counts
of industrial cleanrooms to hospitals as well. These standards are based on the presence
of particles sized 0.3 to 5 µm and are more practical than microbial sampling. Infectious
pathogens, including fungi, bacteria, and viruses, can become airborne in moisture
droplets of 5 to 10 µm after coughing or sneezing. Their small size allows them to
remain airborne for several hours and be carried by drafts for considerable distances.[7]
Currently, the most common laminar-flow system for air filtering and particle control
is the high-efficiency particulate air (HEPA) filter, in which particles 0.3 µm or
larger are removed with 99.97% efficiency.[8] The high efficiency of the HEPA filtering system depends on the complete absence
of leaks, which leads to greater maintenance costs when compared with other types
of filters. Additionally, the efficiency of a HEPA filter will increase as particles
accumulate in the filter. However, this particle build-up will decrease the airflow
rate, in turn, decreasing the number of air exchanges per hour that the unit can supply,
requiring constant monitoring.[7]
An alternative tool that has been studied as a potent sanitizing agent in the fields
of medicine and dentistry, is ozone gas due to its already well-documented antimicrobial
properties.[8] In addition, its therapeutic application has been the subject of numerous studies,
not only for its antimicrobial properties but also for its anti-inflammatory action.
It has been clinically used for wound healing, as a high O2 tension increases the formation of granulation tissue, favoring tissue repair.[9] In addition, it can promote the increased generation of growth factors and activate
local anti-oxidant mechanisms; it also promotes tissue repair.[10]
[11]
Ozone can inactivate microorganisms by damaging their genetic material. For these
reasons, ozone has been widely used for the inactivation of microorganisms.[1]
[12] This gas is easily and cost-effectively produced and is a natural compound that
quickly decomposes to oxygen with a half-life of ∼20 minutes. Also, it can effectively
penetrate all parts of a room, including those of difficult access by conventional
liquids or manual cleaning procedures.[13] Although ozone has proved effective for the in vitro inactivation of a series of microorganisms, including pathogenic bacteria and viruses
of clinical relevance in nosocomial infections, its use in environmental and hospital
disinfection or sterilization is still not widespread.[14]
[15]
Even with vast literature underpinning the effectiveness of ozone gas on microorganisms
regarding viable particles, so far only a few studies have evaluated its action on
non-viable particles. Therefore, our experimental study proposes an investigation
on the effect of ozonation on the number of viable and non-viable particles comparing
a non-controlled and a controlled environment associated with a HEPA filter.
Materials and Methods
Environment Selection and Classification
Two different areas commonly found in health facilities were tested. In group 1, the
non-critical, non-classified, or non-controlled area used in our tests was a 30 m3 room used as an office. In group 2, the cleanroom selected was an N8 ISO 16444 grade
D (Anvisa RDC n17/2010, 2010),[16] laboratory with 60 m3 located at the Immunobiological Technology Institute, within Oswaldo Cruz Foundation.
Ozonation Procedure
The ozone (O3) air ozone generator (Philozon, Balneário Camboriú, SC, Brazil) was used to disperse
the ozone gas in the chosen environments ([Fig. 1]). Using oxygen from environmental air, the generator produces 10 g/h of ozone at
a flow rate of 1560 L/min. Following the manufacturer's instructions, to obtain an
approximate concentration of 15 to 20 ppm volume (ppmv) of ozone in the environment,
the application time was set by the volume of the treated area (30 minutes: 30 m3 and 60 minutes: 60 m3). Ozone output was previously confirmed by DN 800–03, a portable pump-suction ozone
gas detector (Dino Purification Co., LTD, Guangzhou, China). All tests in each area
were performed on three separate and consecutive days. Humidity and temperature were
registered at the time of each test and considered in the final analysis of the results.
Fig. 1 The O3 air ozone generator and microbial air monitoring system (MAS-100).
Environmental Monitoring
For environmental monitoring in this experimental study, active methods of counting
viable and non-viable particles were used in the selected environments before and
after the ozonation procedure.
Active Sampling of Viable Particles
Before and after ozonation, a microbial air monitoring system (MAS-100; Merck, Darmstadt,
Germany) was used for 5 minutes at a flow rate of 100 L/min to count viable particles
in the air ([Fig. 1]). Two air-samplers were positioned in the tested room 60 and 120 cm from the ozone
generator. The environmental air was aspirated through a perforated lid and impacted
onto the surface of growth media (Tryptic Soy Agar) in a 90 mm Petri dish. After the
air sampling procedure, the Petri dishes were incubated at 37°C for 48 hours. Colonies
were counted and expressed as colony-forming units/cubic meters (CFU/m3).
Active Sampling of Non-Viable Particles
Before and during the ozonation procedure, Lasair III 310C (Particle Measuring System,
Colorado, EUA) particle counter was used to count non-viable particles by active sampling
([Fig. 2]). The particle counter was positioned in the center of the tested environment. The
sampling of non-viable particles followed the specific operating procedures and configurations
of the particle counter. The 0.5 and 5 μm size particles were selected for counting
according to the parameters indicated for N8 grade D cleanrooms described in ISO 16444[16]. The particle counter was adjusted to count the number of particles/m3 and with a sampling volume of 0.283 m3 (283 L).
Fig. 2 Lasair III 310C (Particle Measuring System, Colorado, EUA) particle counter.
Statistical Analysis
The results were analyzed by the Mann—Whitney U nonparametric test using Statistica version 13.3 statistical program to assess the
difference in medians before and after ozonation, considering p < 0.05 as significant.
Results
Environmental Monitoring
In all tests, the relative humidity (RH) and temperature were monitored. In group
2, grade D clean room, these parameters were 70% RH and 20°C. In group 1, non-classified
area, temperature averaged at 22°C with 55% to 75% RH.
Active Sampling of Viable Particles
In group 1, 12 samples were collected and in 5 of them, the microbial growth was such
that it was not possible to count CFU for these samples. In three tests, it was possible
to obtain a microbial count pre-ozonation and in 100% of these, a reduction in the
number of colonies was observed after ozonation. In group 2, with the HEPA filtering
system, it was possible to count the number of colonies in all samples and [Table 1] shows a reduction trend in these results. Due to the impossibility of performing
the CFU count in part of the trials, no statistical tests were performed, and the
results were analyzed regarding only the percentage reduction.
Table 1
Viable particles counting (CFU/m3) before and after ozonation
|
Distance from O3 generator
|
Day test
|
UFC before O3
|
UFC after O3
|
Status
|
|
Group 1
|
1 m
|
Day 1
|
38
|
18
|
Reduction
|
|
|
Day 2
|
Uncountable
|
Uncountable
|
Unchanged
|
|
|
Day 3
|
Uncountable
|
134
|
Reduction
|
|
2 m
|
Day 1
|
21
|
19
|
Reduction
|
|
|
Day 2
|
163
|
96
|
Reduction
|
|
|
Day 3
|
Uncountable
|
Uncountable
|
Unchanged
|
|
Group 2
|
1 m
|
Day 1
|
1
|
1
|
Unchanged
|
|
|
Day 2
|
5
|
5
|
Unchanged
|
|
|
Day 3
|
11
|
5
|
Reduction
|
|
2 m
|
Day 1
|
0
|
0
|
Unchanged
|
|
|
Day 2
|
3
|
0
|
Reduction
|
|
|
Day 3
|
Uncountable
|
4
|
Reduction
|
Active Sampling of Non-viable Particles
[Table 2] depicts the results of the average, median, and standard deviation of group 1's
particle counts after ozonation in each of the three days of trials. After the use
of O3 in group 1, an 89% reduction in the median number of 5.0 μm particles was observed,
while the median number of 0.5 μm particles increased 586%. [Table 3] describes the average, median, and standard deviation of group 2's particle counts
after ozonation in each of the three days of trials. After the use of O3 in group 2, a 97% and 100% reduction in the median number of particles was observed
for particles sized 0.5 and 5 μm, respectively.
Table 2
Non-viable particles counting (particles/m3) before and after ozonation in the Group 1
|
Particle size
|
Results before O3
|
|
Results after O3
|
|
Descriptive statistics
|
|
10 min
|
20 min
|
30 min
|
Average
|
RSD*
|
Median
|
|
Day 1
|
|
0.5 μm/m3
|
7.492.816
|
44.957.524
|
68.076.384
|
66.961.584
|
59.998.497
|
22%
|
66.961.584
|
|
5 μm/m3
|
40.798
|
7.376
|
4.459
|
3.895
|
5.243
|
36%
|
4.459
|
|
Day 2
|
|
0.5 μm/m3
|
15.432.208
|
52.731.968
|
64.462.592
|
60.053.872
|
59.082.811
|
10%
|
60.053.872
|
|
5 μm/m3
|
31.667
|
4.655
|
3.599
|
3.425
|
3.893
|
17%
|
3.599
|
|
Day 3
|
|
0.5 μm/m3
|
8.753.473
|
50.018.908
|
60.366.988
|
52.848.396
|
54.411.431
|
10%
|
52.848.396
|
|
5 μm/m3
|
19.223
|
3.405
|
2.363
|
2.176
|
2.648
|
25%
|
2.363
|
Table 3
Non-viable particles counting (particles/m3) before and after ozonation in the Group 2
|
Particle size
|
Baseline
|
Measurements after O3
|
Descriptive statistics
|
|
10
min
|
20
min
|
30
min
|
40
min
|
50 min
|
60
Min
|
Average
|
RSD*
|
Median
|
|
Day 1
|
|
0.5 μm/m3
|
1596
|
21
|
14
|
4
|
7
|
11
|
21
|
13
|
54%
|
13
|
|
5 μm/m3
|
233
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
–
|
0
|
|
Day 2
|
|
0.5 μm/m3
|
1155
|
92
|
53
|
99
|
74
|
56
|
187
|
94
|
53%
|
83
|
|
5 μm/m3
|
102
|
7
|
4
|
7
|
4
|
0
|
21
|
7
|
101%
|
6
|
|
Day 3
|
|
0,5μm/m3
|
1303
|
14
|
25
|
7
|
21
|
28
|
25
|
20
|
40%
|
23
|
|
5μm/m3
|
141
|
0
|
0
|
4
|
0
|
0
|
0
|
1
|
245%
|
0
|
The results found in groups 1 and 2, before and after ozonation were compared using
the Mann–Whitney U nonparametric test. In both groups, it was observed, with a 5% significance level,
that there was a statistically significant difference (p = 0.009 in group 1 and p = 0.002 in group 2) in the number of 0.5 and 5 μm particles before and after the
use of O3 ([Fig. 3]).
Fig. 3 Box plot variation of non-viable particles counting (particles/m3) before and after ozonation.
Discussion
Airborne biological particles, including bacteria, fungi, and viruses, are commonly
found in the breathing air. These microorganisms may serve as respiratory pathogens
and if they are still viable after aerosolization and suspension in the air, they
have the potential to cause respiratory diseases. Therefore, it is important to develop
continued methods for controlling and improving environmental air quality.[1]
[17]
[18]
Monitoring of air quality in healthcare facilities is mandatory. Textile fibers, dust
particles, skin flakes, and respiratory aerosols, for example, can transport microorganisms
and deposit them onto instruments and surfaces of surgical areas, leading to contamination
and potential cross-infection.[5]
[6] A previous study sought to identify the generation and behavior of airborne particles
due to the movement of the medical team when entering and exiting the operating room.
The authors identified that airborne particles were generated from the floor and the
shoes of the medical team. Particularly those sized 0.3 to 0.5 μm were carried up
to the level of the operating table due to the movement of the team in the operating
room.[5]
Currently, the most used method for the reduction of particles in the environment
is the HEPA filter. HEPA filters remove 99.97% of particles 0.3 μm or larger and create
a homogeneous airflow in the operating room with little turbulence. However, they
are expensive to install and maintain and demand continuous monitoring.[19] A previous work correlated airborne microbial contamination with the rate of air
renewal in operating rooms. They concluded that microbial contamination decreased
as the air renewal rate increased.[20] Only a few countries have established acceptable levels of microbial contamination
for operating rooms with conventional ventilation and most recommend 20 air renewals
per hour to get 50 ± 150 CFU/m3 of air.[6]
[20]
In 2018, a study characterized the type and concentration of bioaerosols in an operating
room before and after sterilization and disinfection, finding growth of biological
species even after the cleaning procedures. The authors reported that factors such
as insufficient ventilation, inadequate HEPA filtering, high humidity, and a lack
of proper post-surgical infectious residue management may have been the causes for
the observed increase. These authors suggest designing better ventilation systems,
replacing the HEPA filters, implementing more rigorous and frequent disinfection procedures,
and controlling relative air humidity and room temperature.[21]
Additionally, another work determined the factors that influence air contamination
in a surgical medical center by active sampling methods. They found that even when
the ventilation system was controlled with a HEPA filter, microbial concentration
was far from the ideal levels during surgical activities. Inadequate air filtering
was the main source of contamination. Additionally, the colony count significantly
increased with the increment of 1°C in room temperature and was also correlated to
the number of people in the room.[22]
All these findings led to the search for additional sanitization tools and motivated
us to investigate ozone in this study. Ozone can inactivate microorganisms by damaging
their genetic material. For these reasons, ozone has been widely used for the inactivation
of microorganisms.[1]
[12]
Gaseous disinfectants were introduced in the market as an effective alternative for
manual disinfection.[14] They have also been used as a co-adjuvant sanitization method leveraging the performance
of HEPA filtering. Gaseous disinfectants as ozone have antimicrobial properties four
times greater than liquid disinfectants, due to their uniform distribution and penetration.
Beyond its effective diffusibility, ozone has a broad antimicrobial spectrum. When
compared with chlorine, ozone gas requires a lower concentration and application time
to effectively disinfect.[23]
In a recent study, the authors evaluated the microbial decontamination after an ozone
application within a triage area that receives patients with suspected cases of COVID-19.
Air and surface samples were collected to evaluate the efficiency of ozone for microbial
reduction. The authors concluded that an ozone system within the rooms of a COVID-19
triage area was highly efficient and led to significant reductions (p < 0.05) in bacterial and fungal counts. Furthermore, it is possible to assume that
ozone application could also be useful for inactivating the SARS-CoV-2 virus[23]
Hudson et al, in 2008, develop a practical method to test the anti-viral properties
of ozone in a mobile apparatus that could be used to decontaminate rooms in health
care facilities, hotels, and other buildings. Maximum antiviral efficacy was obtained
after a short period of time in a high humidity (>90% RH) and gas concentration 20
to 25 ppm. All 12 viruses tested, on different hard and porous surfaces, and in the
presence of biological fluids, could be inactivated by at least 3 log10.[24]
Another study analyzed the influence of different species of microorganisms, relative
humidity, and ozone dose on the disinfection of surfaces using this gas. The authors
concluded that the survival of microorganisms and the dose of ozone (ozone concentration
times the exposure time) have an exponential relationship. Furthermore, they found
that ozone was a more effective germicide when RH increased. This might be related
to an increase in free radicals generated from ozone's reaction with water vapor from
more humid environments. Finally, the authors concluded that ozone is highly effective
and is a reliable treatment for contaminated surfaces.[12]
Some factors such as temperature, RH, and turbulence are decisive factors in the dispersion
and dissemination of infectious agents in indoor environments. Therefore, from a methodological
point of view, field studies should try to control these potential variables as well
as include effective ventilation systems. Considering these parameters, our study
included ozonation of a controlled and non-controlled environment, both free of human
activity around the equipment to avoid any air turbulence. Additionally, the temperature
and RH of both the controlled and non-controlled rooms were maintained at ∼20°C and
60% RH throughout the experiment.[2]
The effectiveness of ozone (aqueous and gaseous) as an alternative sanitization technology
to conventional disinfectants in the reduction of microbial contamination of water
and air was already assessed. The authors found that the most effective reduction
in microbial load occurred after 20 minutes of ozonation. They concluded that ozone
treatments were effective against microbial contaminants, reducing the CFU of the
investigated microorganisms, making ozone an extremely promising alternative. Furthermore,
the authors highlighted that due to the room's large volume (85 m3), there was an elevated dispersion of ozone, recommending that the experiment be
repeated in a smaller environment to evaluate the positive impact of ozone on airborne
microorganisms.[14]
Taking into account the authors' suggestion, in our study, ozone was applied in smaller
rooms to minimize gas dispersion. The non-controlled area had footage of 30 m3 and the cleanroom of 60 m3, being ozonized respectively for 30 and 60 minutes, thus obtaining an average of
10 minutes per 10 m3, as recommended by the manufacturer Philozon. Although our results suggested a trend
in reducing the viable particle count (CFU/m3), this is just an initial observation, further tests must be performed on the number
of viable particles, which is one of the limitations present in this study.
However, we were able to quantitatively analyze non-viable particles, which is the
main recommended method for monitoring clean rooms (ISO 16444: 2015).[16] In previous work, methods to monitor and interpret airborne particles in a cleanroom
were compared. The authors concluded that viable and non-viable particles are indeed
correlated, recommending the count of non-viable particles as a routine procedure
in preventing an increase in microbial air contamination.[6] Besides reducing the contamination risk of those manipulating live microorganisms,
this method can also be used as a diagnostic tool for air treatment. Counting viable
particles, even if by an automated process, requires expensive culture media and longer
incubation and interpretation times.[1]
[4]
[20]
In our study, we showed that ozone gas reacts with non-viable particles significantly
reducing the number of 5 μm particles in both tested rooms, non-controlled and controlled
with a HEPA filtering system. Furthermore, our results showed that in the non-controlled
environment, without the HEPA filter, ozone reacted with the particles therein promoting
a substantial decrease in 5 μm particles. However, due to their fragmentation, a significant
increase in the number of 0.5 μm particles was observed[25]
[26] ([Fig. 3]).
These results are in accordance with a previous one that also found the increased
formation of particles after indoor ozonation in a non-controlled room. The authors
explored the effects of ozonation in the presence of a common terpene source in residential
dwellings. The results of their study suggest that the occupants of residential dwellings
may also be exposed to elevated levels of fine and ultra-fine particles when an ozone
generator is employed in a residential setting, particularly during periods of relatively
high terpene concentrations, e.g., during the use of pine oil-based cleaners or scented
deodorizers, what should be a concern with respect to elevated inhalation exposure.[26]
Ozone's reaction with several compounds occurs in two different ways and both coexist.
One involves direct reactions of molecular ozone and the other occurs through reactions
of its subproducts, such as free radicals. The reactions between ozone and some unsaturated
hydrocarbons can be an important source of secondary pollutants, including free radicals,
carbonyl, and carboxylic acids. The formation and effect of these secondary organic
particles in human health need to be clarified.[12]
[26]
In contrast, in the controlled room, this increase in 0.5 μm particles was not observed
as these particles were almost eliminated when ozone was applied in a room with a
HEPA filtering system. These data suggest that the particles cleaved by ozone were
effectively filtered from the environment by the HEPA filter because a significant
reduction in the total particle count (0.5 μm and 5 μm) was observed when compared
with the counts obtained before ozonation, just with the HEPA filter. It is clear
from our tests that ozonation improved HEPA filtering.[22]
[26]
The disadvantage of indoor ozonation is ozone's toxicity to the pulmonary alveoli
when inhaled at high concentrations. For this reason, it cannot be used in constantly
populated areas. This means that ozone can only be used in environments that can be
closed off and emptied during application. Although toxic at high concentrations,
ozone quickly dissociates into oxygen and, if necessary, a catalytic converter can
be used to reverse ozone to oxygen. The protocol used in this study followed the manufacturer's
instructions which suggest that in a 30 m3 room, the ozone generator should remain active for 30 minutes in a room free of people.
Therefore, although this is indeed a limiting factor, it does not impede the use of
ozone.[24]
Conclusions
In this study's conditions, ozone gas was able to significantly reduce the amount
of 5 μm particles regardless of the environment's classification. Also, the HEPA filter
increased the effectiveness in the reduction of 0.5 μm particles. As a limitation
of our study, the results of the active sampling of viable particles could not be
statistically analyzed and were excluded from our conclusion. Considering that particles
5 μm or larger are important in the air transport of microorganisms, their reduction
in the environment is an important factor in controlling cross infections. Ozone gas
has proven to be an effective tool in controlling such infections and can be applied
to a variety of indoor environments, those controlled with a HEPA filter or common
areas such as offices, homes, and hotels. Further investigations into the formation
of secondary organic particles must be conducted.
