SARS‐CoV‐2 indoor environment contamination with epidemiological and experimental investigations

Abstract SARS‐CoV‐2 has been detected both in air and on surfaces, but questions remain about the patient‐specific and environmental factors affecting virus transmission. Additionally, more detailed information on viral sampling of the air is needed. This prospective cohort study (N = 56) presents results from 258 air and 252 surface samples from the surroundings of 23 hospitalized and eight home‐treated COVID‐19 index patients between July 2020 and March 2021 and compares the results between the measured environments and patient factors. Additionally, epidemiological and experimental investigations were performed. The proportions of qRT‐PCR‐positive air (10.7% hospital/17.6% homes) and surface samples (8.8%/12.9%) showed statistical similarity in hospital and homes. Significant SARS‐CoV‐2 air contamination was observed in a large (655.25 m3) mechanically ventilated (1.67 air changes per hour, 32.4–421 L/s/patient) patient hall even with only two patients present. All positive air samples were obtained in the absence of aerosol‐generating procedures. In four cases, positive environmental samples were detected after the patients had developed a neutralizing IgG response. SARS‐CoV‐2 RNA was detected in the following particle sizes: 0.65–4.7 μm, 7.0–12.0 μm, >10 μm, and <100 μm. Appropriate infection control against airborne and surface transmission routes is needed in both environments, even after antibody production has begun.


| INTRODUC TI ON
Increasing scientific evidence indicates the dominance of short-and long-range airborne transmission of SARS-CoV-2. [1][2][3][4][5][6] The observed transmission risks have been higher indoors than outdoors, 7 and discussion on precautions for hospital and home environments has been intense.
In a study that aerosolized SARS-CoV-2 under laboratory conditions, aerosols' infectivity was retained for up to 16 h, 8 while another study estimated the half-life in aerosols to be approximately 1.1-1.2 h (95% CI 0. 64-2.64). 9 Outside of the laboratory, signs of viable SARS-CoV-2 in the air have been detected, [10][11][12] and the virus has also been cultured from exhaled air. 13 A direct link between SARS-CoV-2 viral load, emission, and airborne concentration was recently demonstrated by Buonanno et al. 14 A few studies have detected SARS-CoV-2 RNA in the air at home-environment. 15,16 In hospitals, PCR-based studies have found SARS-CoV-2 RNA in room air, [17][18][19][20][21][22][23][24][25] as well as from air conditioning filters located over 50 m from the patient room. 26 Even though previous studies have mainly used long collection times or high flow rates, challenge is that only a proportion of the air present in a room can be analyzed.
Additionally, indoor turbulence highly affects local concentrations. 27,28 Thus, questions remain about the risk of infection during shorter meetings or in rooms with a larger air space, and whether the findings would be similar in the home environment. As environmental sampling is highly demanding and sample sizes rather small, more patient data are also needed to draw further conclusions in future systematic reviews.
According to laboratory studies, the stability of SARS-CoV-2 on surfaces varies depending on the surface type and environmental conditions. 9,[29][30][31][32] However, its ability to sustain infectivity on surfaces outside laboratory conditions is largely unknown. 33 SARS-CoV-2 RNA has been found, for example, on high-touch surfaces, floors, and toilets, 18,19,34,35 and there are a few possibly positive culture findings of SARS-CoV-2 from the surfaces. [36][37][38] The effect of age and neutralizing antibodies (NAbs) on the spread of SARS-CoV-2 has been speculated, 39-41 but there is a lack of clear evidence for the role of patient-related factors.
This study sought to increase knowledge of SARS-CoV-2 transmission in different environments by analyzing air, surface, and patient samples from a COVID-19 cohort ward in Helsinki University Hospital (HUS), Finland, and from patients' homes. The aims were to determine whether SARS-CoV-2 RNA or viable virus could be found in the home and hospital environments, and which patient-and environment-related factors affect the risk of environmental contamination. A team consisting of researchers from HUS, the University of Helsinki, the Finnish Meteorological Institute, and the Finnish Institute of Occupational Health was established to enable a multidisciplinary approach to the above research questions.

| Index patients and safety measures
Patients were voluntary participants with a qRT-PCR-confirmed symptomatic COVID-19 infection between 1.7.2020 and 16.3.2021.
None of the participants had been vaccinated. As infectivity has been observed to be highest in early disease, the patient with the most recent onset of symptoms was selected as the index patient, 42 except for collection 13, where all the patients in the room had been symptomatic for over 10 days and the patient with the freshest positive PCR result (P26) was selected (Table S1). Environmental measurements were performed in the vicinity of the index patients.
Saliva samples were also collected from other patients who were in the ward at the same time and who agreed to the study in 4 collections (named as "other"). Family members of the home patients were examined for infection and seroconversion. Environmental sampling was performed twice with patients P2 and P3, and P2 was considered as an index due to the more recent start of the symptoms; however, some personal items from both were sampled. The sampling process is presented in a flow chart in Figure 1.
All research personnel conducting the sampling followed aerosol safety protocols and precautions and no infections were detected.
All procedures that involved human participants, including environmental sampling, were conducted in accordance with the ethical standards of the institutional or national research committee and the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The Ethics Committee of Helsinki University Hospital approved the study protocol (HUS/1701/2020). All respondents provided written informed consent prior to their participation.

| Infection prevention protocols in the hospital
The infection prevention and control protocols on the COVID ward included hand hygiene, universal masking for staff (FFP2/3 for ICU and surgical masks for the COVID ward), guidance on social distancing (2 m), and personal protecting equipment (PPE) following droplet precautions. The patients did not use face masks. The ward and

Practical implications
• The finding of SARS-CoV-2 RNA from the air in the absence of aerosol generating procedures (AGP) and in the absence of respiratory symptoms emphasizes the use of respiratory protection and airborne precautions also in situations where AGPs are not performed and regardless of the patients' symptoms.
• Families that used respiratory protection were able to prevent further infections.
• Air and surface contamination was detected in both homes and hospital even though the day from the start of the symptoms was later in hospital measurements. This may follow from more severe disease and increased viral loads which were associated with older age. Infection control measures should be used in both environments to prevent further infections.
ICU were cleaned twice a day between 9 to 10 am and 4 to 5 pm.
The sample collections were done between cleaning around 11 am to 9 pm and thus reflect quite reliably patients' infection status of the collection day. The specific cleaning protocol is presented in the supplement.

| Cell lines
Vero E6 cells (VE6) and their TMPRSS2-expressing clone VE6-TMPRSS2-10 (VE6T) 43 were grown as previously described. 44 To inhibit fungal growth, 0.205 μg/ml of amphotericin B (Fungizone, Thermo Scientific) was added to the medium of the cells that were taken to the hospital for aerosol collections. The used VE6 cell line is originally from ATCC (American type Culture Collection 45 ), and the VE6T cell line has been modified from the original line according to the previous study. 43

| Sampling protocols for air sampling
Seven different air collection methods were used. Details of the collections and samples are presented in Table S3. Sampling times and air volumes varied between different sampling methods depending on the expected optimal collection time for each device according to the manufacturer and previous studies, and the knowledge gathered during the study. A Dekati PM10 cascade impactor (20 L/min air flow, model PMS-420) with three stages (>10.0 μm, 2.5-10 μm, and 1.0-2.5 μm), including a backup filter for particles <1 μm, was used in 11 collections. The three impaction stages were fitted with 25-mm-diameter cellulose acetate membrane filters (CA filter, GE Healthcare Life Sciences) and the backup plate with a 40-mm CA filter. Analyzing the three stages and backup filter, particle distribution according to aerodynamic size (PM10, PM2.5, and PM1) can be ascertained. The collector was placed within 1-2 m from the patient, and particles were collected for 2-4 h. After sampling, filters were immediately placed in 2 ml (25-mm filter) or 3 ml (40-mm filter) of minimal essential Eagle's medium (MEM, Sigma-Aldrich).
The BioSpot 300p bioaerosol sampler prototype (Aerosol Devices Inc.) has a flow rate of 8 L/min and a mechanism that allows water to condense on aerosol particles from as small as 5-10 nm to 20 μm in diameter and minimize the stress when the sample is impacted onto the surface with the collection medium. To increase the sample collection rate, the biosampler is equipped with eight wicking tubes fitted with three nozzle jets to secure gentle transfer of the sample. This sampler was used in 8 collections for 1.5-4 h within a distance of 1-2.5 m from the patient, and the sample was collected in 1-2 ml of MEM.
As a more portable solution for personal area air sampling, a standard 25-mm gelatin (Sartorius Stedim Biotech) or mixed cellulose ester (MCE) filter equipped in the Button sampler with a Gilian F I G U R E 1 Patient inclusion and sampling process and additional analysis performed in the study 5000 air sampling pump, 4 L/min air flow, and a porous curved surface inlet was used in 9 collections. The Button sampler collects particles smaller than 100 μm. 46 The stability of SARS-COV-2 on two filter materials was compared under laboratory conditions to select the more optimal filter type and to optimize the collection time (details in Supplementary Material). Samples were collected for 10-30 min from patients' breathing area. Depending on the health status, a conversation was prompted to increase the output of aerosols. The collection filter was removed into 3 ml of MEM immediately after collection ended.
Three Andersen cascade impactors (400 W pump and 28.3 L/min flow rate) were used simultaneously in six collections. The impactors consist of six stages with size cut points of (1) >7 μm, (2) 4.7-7.0 μm, An additional inlet was used during measurements limiting the upper limit of the particle size to 12 μm. To ensure the correct volume flow rate, each Andersen impactor was fitted with a TSI flow meter. To evaluate the real-time particle number concentration during the hospital collections and to gather additional air samples, a Dekati eFilter was used in two collections. The eFilter monitors changes in real-time particle concentration by utilizing a small diffusion charger powered by an inner chargeable battery. The charge changes were automatically translated into a signal, which was recorded on a data card. When postprocessing the data, the raw charge signal was further converted to represent particle number concentrations using a conversion factor (411 cm −3 fA −1 ) provided by the manufacturer. A count median diameter (CMD) of 60 nm and a geometric standard deviation (GSD) of 1.5 were assumed. 47,48 In addition, the eFilter simultaneously collected samples on a 47-mm gelatin filter using an external pump. After sample collection, the gelatin filter was transferred into 6 ml of MEM. The eFilter was fitted with the same EPA-designed inlets as the Andersen cascade impactors. The Living cells were transported to the laboratory in a warm environment with heat accumulators warmed to 37°C. One plate was used as a negative control to ensure that the cells survived the transport. Other samples were transported with cold accumulators and handled during the same or next day.

| Sampling protocols for surface sampling
Altogether, 252 surface samples in 26 collections for qRT-PCR testing were taken from surfaces in possible direct or indirect contact with the patient (Table S3)

| Other sampling protocols
Saliva samples were taken from 26 index patients either with a Dacron swab (collections 5-9) or by spitting into a Falcon tube (from collection 10 onwards). Ten additional saliva samples were collected from other patients from the ward in four collections and from seven healthy family members of home-treated patients. If possible, patients were asked to rinse their mouth before sampling. In collection 23, the index patient and a healthy family member also took followup saliva samples until 12 days from the start of the patient's symptoms. In collection 26, follow-up saliva samples were taken from patients until Days 14-17 from the start of symptoms.
Nasopharyngeal samples from consenting patients were taken and sent to HUSLAB for a fresh diagnostic PCR. 49,50 Serum samples from consenting patients were taken within a day from sampling and tested for SARS-CoV-2 IgG antibodies with two different tests. 51 Serum samples (dilutions 1:10 to 1:640) were studied with the microneutralization assay. 52 Blood lymphocyte and eosinophil counts, and plasma CRP from consenting patients were measured within a day of sampling, and plasma ferritin, ALP, ALT, D-dimer, and fibrinogen levels within 3 days. The respiration rate and SpO2 levels were measured during the same day (Table S1).
Since the first cases caused by variants of concern (VoC) were detected in Finland at the end of December 2020, they were determined from all patients as a part of routine diagnostics. This information was used to compare the results between VoC strains (mainly alpha in Finland) and non-VoC strains. Virus strains of collections 1-22 (P1-P45) were considered as non-VoC, as they were collected before the first cases were reported in Finland.  were analyzed with the thermocycler software Rotor-Gene 6.0.31 (Qiagen) using similar criteria as with other samples described above.
A 200μl sample of culture medium was taken from those samples that had unclear results based on microscopic observation or possible CPE and tested with N Charité qRT-PCR. Culturing was considered positive if CPE was detected and the Ct value of qRT-PCR performed from the culture media was under 20. If Ct value was higher, it was judged to be caused by original (possibly noninfectious) virus in the sample instead of virus growth. All virus culturing was performed in a BSL3 laboratory.
Optimization of the culturing protocols is described in more detail in the Supplementary Material.

| Statistical tests and design
Statistical tests were carried out with SPSS IBM Statistics version 27.
When comparing means between two independent groups, data were first tested for normality with the Shapiro-Wilk test before testing them either with the independent-samples t-test or a non-parametric test (independent-samples Mann-Whitney U-test for two groups and independent-samples Kruskal-Wallis test for more than two groups

| Patient characteristics and collection surroundings
We
The samples were divided into actively and passively collected samples  Table S2). Estimated copy numbers varied between 1.04 × 10 3 copies/ml and 2.05 × 10 7 copies/ ml. All air samples were cultured, but no viable viruses were observed.

| Effects of patient factors on environmental contamination
Positive air samples were found even when the index patient did not report any respiratory symptoms (2/3, 66.6%). However, there was a statistically significant connection between low oxygen saturation (SpO2) levels and SARS-CoV-2 RNA findings from surfaces, and a possible but nonsignificant connection between low SpO2 levels and RNA findings from the air (surface: p = 0.026, air: Table S2). Toilet surfaces were qRT-PCR positive in 33.3% (3/9) of cases when the index patient had GI symptoms and 0% of cases (0/9) when the index patient did not report any GI symptoms (p = 0.229, Table S2). No positive environmental samples were obtained if the saliva sample from the index patient was negative with both qRT-PCRs. Positive surface samples were detected more often when there were multiple COVID-19 patients in the ward/house during the sampling (p = 0.018, Figure S1). However, no statistically significant difference was detected for air collections (p = 0.845) ( Figure S1). Possible but statistically nonsignificant associations were observed between positive environmental samples and an earlier symptom day, as well as an older age ( Figure S1 and S1c).
No statistically significant connections were found between air and

| Transmission of COVID-19 to family members
The spread of COVID-19 within the family was examined by collecting saliva samples from family members of the five home-treated patients and analyzing qRT-PCR results and SARS-CoV-2 antibody levels. In two families that used protective measures, including respiratory protection (surgical mask or respirator) and intensified cleaning, no further infections were detected. One of these families used masks in common areas, but not in their own rooms behind closed doors. In another family, the bedroom was shared, and masks were used all the time. However, in three families that did not apply

| DISCUSS ION
This study detected considerable SARS-CoV-2 RNA contamination from both home and hospital environments. The virus was found in the air in particle size ranges of 0.65-4.7 μm, 7.0-12.0 μm, >10 μm, and < 100 μm in diameter (Table 2), supporting existing literature. 17,19,20,22 Our findings also support discoveries that normal respiratory activities generate infective particles even in the absence of AGPs, 2,6,64,65 and respiratory symptoms. Additionally, low oxygen saturation showed a connection with a higher possibility of SARS-CoV-2 surface findings and a potential connection with air findings, which could follow from increased particle generation due to respiratory stress. Most (83%, 15/18) of our positive air samples with known particle size were in particles smaller than 4.7 μm, which supports the findings that at least 85% of the viral load is emitted in aerosols smaller than 5 μm. 6,20,66 This is in line with the fact that particle generation produces a distribution which form depends on the activity that is causing the particles. In human respiratory activities, generated particles are mainly small, under 5 μm in dry size distribution. 67,68 A previous study showed that a high sampling flow rate increases the success rate in detecting SARS-CoV-2 from air samples. 21  only qRT-PCR findings, support concerns that a shorter exposure time should be considered, at least for close contacts. 70 Overall, the exposure risk is cumulating with time and no limit to zero risk can be determined. The risk for infection depends on the concentration exposed to (depending on ventilation and produced quanta) as well as persons immunity. 27 when there were only two patients. Overall, larger spaces are considered safer than small ones due to the larger air volume per person. 81 However, it seems that also larger indoor spaces may form a risk environment if occupied by an infected person for a prolonged time period. [82][83][84] In our study, all patients in the ward were COVID patients. However, in many countries, COVID-positive patients have been separated from COVID-negative ones with just curtains and distance. As hospital-acquired infections have been a significant part of overall infections and deaths, [85][86][87] it is important to reduce the risk of infections in hospital wards. It should be noted that the infective aerosol particles may still generate an infection risk even when larger space allows more dilution with increasing distance, as shown by Karan et al. 88 Our findings were mainly from a close distance similar to a previous study that saw higher probability to environmental findings inside 2-meter range, 24 even though the risk for infection especially in prolonged exposure remains also further away. 27,28 It is interesting that the proportion of the positive samples was similar in hospital and home even when the ventilation was more efficient in the hospital and patients have later symptom day. This may be due to more patients in the same room or higher overall viral load which has been associated with more severe disease and higher age in previous studies. 89,90 We observed a trend for an older age being associated with a higher viral load ( Figure S5a) and a larger number of positive surface samples but confirming this would require further studies with a larger sample sizes. In earlier studies, higher viral loads have been associated with an increased probability of viral transmission. 91,92 Possible reasons for the relationship between age and infectivity include reduced saliva production, differences in mucus viscosity and salivary immunoglobulins, 93  Other more frequently qRT-PCR-positive surfaces included highly touched personal items, hospital equipment, and the floor, which is in line with the previous findings. 18,19,34 Even though RNA may persist on surfaces for some time, RNA findings most likely result from contamination on the same day due to daily cleaning.
The building body of evidence supports airborne route predominance for SARS-CoV-2 transmission, [1][2][3][4]14 and an animal study indicates that aerosol inoculation is a more efficient route and causes more severe pathology and higher viral loads. 100 Fomites have not been proven to serve as the sole or primary vehicle of transmission. 101 The probability for surface transmission is estimated to be likely rare, generally less than 1 in 10 000, and the disease manifestation milder. 102 The environmental samples that commonly presented infectious virus in previous studies were mainly in direct contact with infected patients' mucus membranes, or saliva or sputum secretions (e.g., nasal prongs, nasal canula, used tissue, patients mask, and endotracheal tube). 25,38 In this study, families that took protective measures (including isolation of the infected family member) and respiratory protection (surgical masks or FFP2 respirators) were able to prevent further infections even when qRT-PCR-positive samples were collected from both surfaces and air. However, in a household where all surfaces were cleaned many times a day but no respiratory protection was used, all family members became infected. This supports the importance of air hygiene, including also portable air cleaners as a supportive method as shown in previous studies, 15,28 and also encourages control of infection spread in homes. Similar findings supporting the use of masks, isolation with closed door, and opening windows in home environment were found to lower the risk of contamination in the work of Picard et al. 103 Overall our results indicate that transmission may happen through several transmission routes as supported also in previous systematic review. 20 Infection control is even more important with VoC strains that feature a higher rate of household transmission. 104 To better understand the infectivity and state of the infection compared to the environmental findings, we collected saliva and serum samples. SARS-CoV-2 was cultured from saliva during symptom Days 2-11. 42 SARS-CoV-2 RNA was detected in the saliva of patients who had already formed IgG and NAbs, which align with previous findings of prolonged RT-PCR-positivity. [105][106][107] In addition, the saliva of P16 on symptom Day 11 was still positive in virus culture, even though the patient had NAbs. Moreover, we obtained positive air and surface samples when the index patient had a positive IgG result and NAbs, which agrees with the findings of Lei et al. 108 This contradicts the suggestion that NAbs solely could be a reliable marker for non-infectivity 78. In the view of infection control, we agree with Lei et al., 108 Tan et al. 109 and Wölfel et al. 110 78 suggesting that the results from the earlier variants can still be considered to provide valid information for the current situation.
Our study also has some limitations. Even though the overall number of our samples is quite high compared to previous studies, it is still limited in the statistical aspect and only able to detect major differences and associations. As environmental sampling is time consuming and resource intensive, making it challenging to achieve a statistically large enough sample size, it is important to combine findings from several different studies for more detailed analysis.
We only conducted environmental sampling at a single time point. In the future, a longitudinal examination could enable a more accurate examination of the effects of the course of disease for environmental contamination. The mean time from the onset of symptoms until sampling varied between homes and hospital and may affect the results. However, as the infection requiring hospital treatment is more severe, the viral loads may stay high longer, 112,126 and accordingly, no major differences were detected in viral load in saliva in our study between homes and hospital patients. This would provide rather similar expectation for environmental contamination as Buonanno et al examined. 14 Also, patients generally arrive to the hospital at the later stage of the disease (excluding hospital-acquired infections that were not detected in this dataset) which makes our dataset suitable to represent the real situation between homes and hospitals.
In addition, we only measured the IgG and NAb response, but viral secretion from mucus membranes can continue if the IgA response is weak. 127 The IgA immune response should thus be examined further in upcoming studies. The qRT-PCR results might include some uncertainty due to the differences in the texture and fluidity of saliva and should be considered as estimates. As many samples were collected from a large patient hall, it is possible that some observed viruses might have originated from other than the index patient.
However, most of the surface samples were from patient-specific surfaces, and aerosols are known to concentrate near the source, 71 indicating that most of the positive samples are expected to be produced by the index patient. Particle size cutoffs in Andersen samplers might be slightly higher than estimated, as the amount of liquid used in the sampling was slightly smaller than recommended due to practical reasons. Finally, we strongly suggest developing a new, more sensitive methodology for assessing the virus viability to better assess transmission mechanisms.

ACK N OWLED G M ENTS
We thank Esa Pohjalainen for assistance in the laboratory, Emma Klemetti for the illustrations of

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.