HEPA filters of portable air cleaners as a tool for the surveillance of SARS‐CoV‐2

Abstract Studies about the identification of SARS‐CoV‐2 in indoor aerosols have been conducted in hospital patient rooms and to a lesser extent in nonhealthcare environments. In these studies, people were already infected with SARS‐CoV‐2. However, in the present study, we investigated the presence of SARS‐CoV‐2 in HEPA filters housed in portable air cleaners (PACs) located in places with apparently healthy people to prevent possible outbreaks. A method for detecting the presence of SARS‐CoV‐2 RNA in HEPA filters was developed and validated. The study was conducted for 13 weeks in three indoor environments: school, nursery, and a household of a social health center, all in Ciudad Real, Spain. The environmental monitoring of the presence of SARS‐CoV‐2 was conducted in HEPA filters and other surfaces of these indoor spaces for a selective screening in asymptomatic population groups. The objective was to limit outbreaks at an early stage. One HEPA filter tested positive in the social health center. After analysis by RT‐PCR of SARS‐CoV‐2 in residents and healthcare workers, one worker tested positive. Therefore, this study provides direct evidence of virus‐containing aerosols trapped in HEPA filters and the possibility of using these PACs for environmental monitoring of SARS‐CoV‐2 while they remove airborne aerosols and trap the virus.

Moreover, the use of face masks was mandatory in indoor and outdoor spaces at school except for preschool children. 4 Until June 23, 2021, a total of 260 699 children up to 9-year-old (around 7.5% of the total confirmed cases) were confirmed as SARS-CoV-2 positive in Spain. 5 The COVID-19 transmission is more likely indoors than outdoors which can be explained by airborne transmission through the presence of aerosol containing viable viral particles, generated by infected people. 6 This especially occurs in closed and poorly ventilated spaces. [7][8][9][10][11] Viable Delta variant SARS-CoV-2 has been detected in air collected by air samplers located ~3 m from an infected individual experiencing mild symptoms as well as through surface sampling of a mobile phone that was in frequent proximity to the individual's exhaled breath. 12 The SARS-CoV-2 containing particles with diameters <100 μm can float in the air from minutes to several hours. 13 For this reason, natural or mechanical ventilation is highly recommended in indoor environments to reduce the risks of virus transmission. However, natural ventilation depends on weather conditions and building structure, which make it difficult to achieve a high enough ventilation rate at all time. In those cases, the use of portable-filter air cleaners (PACs) can be an effective supplementary measure to improve the cleaning of indoor air contaminated with virus-containing aerosols. PACs are used to remove particulate matter from indoor air and a very common particulate filter is the high-efficiency particulate air (HEPA) filter, in which the single-pass efficiency of the filter media is >99.97% for 0.3 μm particles (Most penetrating particle size) with an equal or higher efficacy for other aerosol sizes. 14 As measured by Liu et al., 15 the peak concentration of SARS-CoV-2 aerosols appears in two distinct size ranges, one between 0.25 and 1.0 μm, and other larger than 2.5 μm. The filtration efficiencies of HEPA filters, employed in most air purifiers is high enough to remove this size range of potentially infectious aerosols.
The analysis of dust collected on filters in central forced-air heating, ventilation, and air conditioning (HVAC) or portable systems to determine the presence of biotic and abiotic contaminants in indoor air have been widely investigated and are collected in Haaland and Siegel's review. 16 Several experimental studies have provided evidence for the potential of portable HEPA purifiers to eliminate airborne SARS-CoV-2. [17][18][19][20][21] Rodriguez et al. 20 analyzed the indoor air of positive patients in households before and after using a PAC. All the air samples collected (by using an MD8 Airport portable air collector with gelatin membrane filter) before using PACs were positive for SARS-CoV-2 while after switching on PACs, all samples except one were negative, but authors highlighted the fact that in the negative case the sampled room size was higher than recommended for the use of this device. In addition, the manufacturer of PAC used did not provide the clean air delivery rate (CADR), an important metric used to characterize air cleaners whose units are cubic meter per hour. On the contrary, Curtius et al. 22 tested the efficiency and practicability of PACs in a high school classroom while regular classes were taking place. They monitored the aerosol number concentration for particles >3 nm at two locations in the room, the aerosol size distribution in the range from 10 nm to 10 μm and particulate matter PM 10 with windows and door closed, concluding that the aerosol concentration was reduced by more than 90% within less than 30 min when running the PACs. Morris et al. 23 detected SARS-CoV-2 in a COVID-19 ward before activation of HEPA-air filtration and also after filter deactivation but not during filter operation. SARS-CoV-2 RNA was evaluated in the various size fractions of air samples collected using National Institute for Occupational Safety and Health (NIOSH) BC 251 2-stage cyclone aerosol samplers. Air filtration simulation experiments carried out by Ueki et al. 24 quantitatively showed that an air cleaner equipped with a HEPA filter could continuously remove SARS-CoV-2 from the air.
During pandemic, HEPA filters have also been used as sampling system to test the presence of SARS-CoV-2. 19,25,26 Burgos-Ramos et al. 19 analyzed HEPA filters exposed for 3 months in a waiting room and three treatment rooms from dental clinics. SARS-CoV-2 was detected in the filter of the waiting room but not in the treatment rooms. They concluded that the use of H 2 O 2 solution (1%) for 1 min for mouth rinsing drastically reduced the possibility of SARS-CoV-2 spread during aerosol-generating dental procedures. Recently, Zuniga-Montanez et al. 25   and precautionary measure to reduce SARS-CoV-2 transmission, especially in indoor spaces, where many people are gathered such as classrooms. Additionally, PACs with HEPA filters are also recommended as a measure to improve indoor air quality. [27][28][29][30] Regarding the viral infection, there is strong evidence that respiratory transmission is dominant, with limited impact of transmission

PRACTICAL IMPLICATIONS
• A method for detecting SARS-CoV-2 RNA in HEPA filters was developed and validated.
• HEPA filters tested positive for SARS-CoV-2 in two school classrooms despite the mandatory use of masks.
• Direct evidence of virus-containing aerosols trapped in HEPA filter is provided.
• The school and nursey classrooms studied were negative for SARS-COV-2 RNA, both for the HEPA filter and surfaces, consequently COVID-19 cases were not notified.
• The positive HEPA filter for SARS-COV-2 RNA found in an indoor space allowed to find an infected worker. by surfaces. 31 Nevertheless, surfaces can be used for viral monitoring due to the permanence of the virus on them, which can be useful as a surveillance tool. [32][33][34] For example, the detection of SARS-CoV-2 RNA in air filters or by means of sponges on surfaces 35 indicates the current or recent circulation of viruses in the space investigated, although the presence of viral RNA is not an indication of infectivity.
Based on these premises, the main objective of this study was to employ environmental monitoring of the presence of SARS-CoV-2 in HEPA filters and other surfaces in indoor spaces in which ventilation strategies are used to avoid virus transmission. The study was conducted for the selective screening in asymptomatic population groups in a school, a nursery, and a household of a social health center to identify and try to limit outbreaks at an early stage in these institutions of interest for Public Health. To approach this objective, a method for determining the presence of SARS-CoV-2 RNA in HEPA filters was developed, optimized, and validated under realistic conditions where PACs were running, and people infected with COVID were notified subsequently. Later, a monitoring campaign was conducted for 13 weeks in the three indoor environments.

| Portable Air Cleaners Characteristics and Operation
The tests were performed with commercially available PACs (Winix Standard. 36 The volume flow through the Winix Zero can be adjusted in five stages: sleep, low, medium, high, and turbo. According to the manufacturer the CADR for each stage is 60, 120, 196, 270, and 390 m 3 /h, respectively. The device is designed for rooms of up to 99 m 2 . In the case of Winix Zero Pro, it also presents five stages; sleep, low, medium, high, and turbo being the CADR 120, 122, 284, 330, and 470 m 3 /h, respectively, and it is designated for rooms of up to 120 m 2 . It is important to highlight that the room size specified by the manufacturer is a recommendation given to reduce the allergens or particulate matter from air and not to reduce SARS-CoV-2. Both PACs have been recommended to be used in stage "high" or "turbo" to reduce the risk of aerosol transmission, the CADR should be set as high as possible. The problem that presents the "turbo" mode is the noise and thus most of the time it is used at stage "high". Table 1 summarizes the characteristics of operation of air cleaners used in this study. The PACs were running during the school lessons from the beginning to the end of lessons (about 5 h) every day, 5 days per week. In the nursery about 7 h per day, 5 days per week, while in the case of the household of the social health center, it was running continuously in the living room.
Regarding filtration characteristics, the air cleaners present a washable pre-filter for coarse dust (made of fine mesh), an active charcoal filter for volatile organic compounds and the glass microfiber HEPA filter H13 for submicron and ultrafine particles. The air cleaners present a patented PlamaWave Technology® that produces OH radical with the aim of removing biological pathogens. However, due to OH radicals can initiate chemical oxidation indoors, leading to a wide variety of chemically complex and unhealthy products, this technology has been recommended to be deactivated. 37  The rest of ACH until 5 or 6 must be achieved by opening windows (ACH ventilation = ACH objective -ACH aircleaner ).

| Sponges
Surface samples were collected using Dry-Sponges (

| Methodology for the determination of SARS-CoV-2 in HEPA filters and sponges
The procedure for the analysis of sponges in the laboratory has been described previously. 35

| Design of the sampling campaign and measurement sites
The environmental surveillance of SAR-CoV-2 has been conducted in a school, a nursery, and a household of a socialhealth center in Ciudad Real (Spain). The main inclusion criteria to take part in the study was to be part of asymptomatic population groups without known active or recent positive cases. The sites included are:

| Validation of the methodology in the HEPA filters
The validation of the methodology for the detection of SARS-CoV-2 RNA in the HEPA filters was carried out before starting the sampling campaign. On one hand, three PACs with HEPA filters running in two classrooms of different schools (school A and B) and the living room of a household of the social health center, were analyzed after knowing that there were people infected with COVID. Classrooms were closed for 2 weeks and in the case of the household, the infected residents were most part of time isolated in their bedrooms except those who were hospitalized. In these cases, the PACs were running for several weeks before the first positive individual was confirmed.
The classrooms and the household were different from those included in the sampling campaign and described in section 2.4.
The Ct values obtained in the SARS-CoV-2 RT-PCR for the validation of the methodology in HEPA filters are summarized in Table 3. Figure 1A shows    On the contrary, one blank sample (an HEPA filter new without having been exposed to indoor air) for each PAC model, was processed in the laboratory in the same way as sampled HEPA filters.

TA B L E 3 Ct results obtained in the SARS-CoV-2 RT-PCR for validation of the methodology for the detection of HEPA filters
Blanks were negative for SARS-CoV-2 RNA by PCR.

| Environmental surveillance of SARS-CoV-2 RNA in indoor environments
Before starting the environmental surveillance campaign, the HEPA filters were changed in all the studied places, as soon as all the permits were received from the competent authorities.
The results obtained during the study for the surveillance in the It is important to mention that the monitoring campaign started as soon as all permits and authorizations were received from the competent authorities, but it was at a time of the pandemic when the third wave, after Christmas, has already passed with a cumulative incidence in the region of 41 cases per 100 000 inhabitants. 43

| CON CLUS IONS
The studies conducted up to date have measured SARS-CoV-2 in rooms (mainly hospitals but also in nonhealthcare environments) where people were already infected with SARS-CoV-2. By contrast, herein we investigated the presence of SARS-CoV-2 in HEPA filters housed in PACs running in places of great interest for public health with apparently healthy people before anyone had symptoms or tested positive with the aim of trying to prevent a possible outbreak.
A methodology for the detection and surveillance of SARS- the study, and especially thank all the participants in the study (children and residents, their tutors, teachers, and residence workers) for their generosity in being part of this pilot project.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
The study has the definitive approval of the Drug Research Ethical