key: cord-0877219-1n4dastt
authors: Mallach, G.; Kasloff, S. B.; Kovesi, T.; Kumar, A.; Kulka, R.; Krishnan, J.; Robert, B.; McGuinty, M.; den Otter-Moore, S.; Yazji, B.; Cutts, T.
title: Aerosol SARS-CoV-2 in hospitals and long-term care homes during the COVID-19 pandemic.
date: 2021-06-01
journal: nan
DOI: 10.1101/2021.05.31.21257841
sha: 423fdb2366d73e16535ebf6c4dd1a2fe194bf787
doc_id: 877219
cord_uid: 1n4dastt

Background: Few studies have quantified aerosol concentrations of SARS-CoV-2 in hospitals and long-term care homes, and fewer still have examined samples for viability. This information is needed to clarify transmission risks beyond close contact. Methods: We deployed particulate air samplers in rooms with COVID-19 positive patients in hospital ward and ICU rooms, rooms in long-term care homes experiencing outbreaks, and a correctional facility experiencing an outbreak. Samplers were placed between 2 and 3 meters from the patient. Aerosol (small liquid particles suspended in air) samples were collected onto gelatin filters by Ultrasonic Personal Air Samplers (UPAS) fitted with <2.5{micro}m (micrometer) and <10{micro}m size-selective inlets operated for 16 hours (total 1.92m3), and with a Coriolis Biosampler over 10 minutes (total 1.5m3). Samples were assayed for viable SARS-CoV-2 virus and for the viral genome by multiplex PCR using the E and N protein target sequences. We validated the sampling methods by inoculating gelatin filters with viable vesicular stomatitis virus (VSV), and with three concentrations of viable SARS-CoV-2, operating personal samplers for 16hrs, and quantifying viable virus recovery by TCID50 assay. Results: In total, 138 samples were collected from 99 rooms. RNA samples were positive in 9.1% (6/66) of samples obtained with the UPAS 2.5{micro}m samplers, 13.5% (7/52) with the UPAS 10{micro}m samplers, and 10.0% (2/20) samples obtained with the Coriolis samplers. Culturable virus was not recovered in any samples. Viral RNA was detected in 10.9% of the rooms sampled. There was no significant difference in viral RNA recovery between the different room locations or samplers. Method development experiments indicated minimal loss of SARS-CoV-2 viability via the personal air sampler operation. Key Findings: Although a subset of aerosol samples exhibited detectable SARS-CoV-2 RNA at low titres, the presence of viable SARS-CoV-2 virus in aerosols appears to be infrequent at >2m distance.

remains unclear, though it is likely to play a role in super-spreading events. [8, 21, 22] Moreover, 64 modelling studies show that even during close contact interactions, aerosols, rather than larger ballistic 65 droplets, drive viral exposures, as droplets are relatively unlikely to land on a mucus membrane, 66 compared to the higher likelihood of inhaling aerosols. The size of a virus-containing particle may also affect infectivity. One study demonstrated that 93 macaques infected by the aerosol route had more severe disease compared to the 94 intratracheal/intranasal route.

[33] Submicron particles are able to penetrate more deeply into the lungs, 95 depositing in the alveolar region where immune responses may be evaded. [15] Also, in the alveolar 96 region the ACE-2 receptors that the SARS-CoV-2 virus binds to are more accessible, possibly increasing 97 the likelihood of infection. [15, 19] In a subset of locations, we also deployed a Coriolis µ Biosampler (Montigny-le-Bretonneux, France) at 129 an inlet velocity of 150 LPM for 10 minutes (1.5m 3 sample volume). Coriolis air samples were collected 130 into sterile cones containing 5mL of VTM, which was reduced to 3mL by evaporation during sample 131 collection. The Coriolis sampler collects particles larger than 0.5μm in size, with no specified cutoff. 132 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. For qualitative detection of viable virus present (safety testing), media from previously seeded 6-well 163 plates was aspirated and 4 mls of fresh VCM was added. To this, 500ul of sample was added and 164 incubated at 37°C +5% CO2 for 5-7 days. Wells were examined, compared to a negative control for CPE, 165 and scored positive or negative based on evident CPE in the cell monolayer. 166

Method validation using surrogate virus (VSV): 167 We evaluated the ability of the gelatin filters to maintain virus viability following a 16-hour sampling 168 period. Vesicular stomatitis virus (VSV) was diluted to 10 5 -10 6 TCID50 /ml in a tripartite soil load and 169 spotted onto gelatin filters over 5 different spots in 2ul volumes.

[39] Three filters were used as a 170 positive control and processed immediately, three others were kept overnight in the Biosafety Cabinet 171 (BSC), and three gelatin filters were placed in a UPAS sampler where it was left running for 16 hours. The 172 following morning, we inoculated an additional 3 gelatin filters and set a sampling time for 4 hours. 173

After sampling times were complete, 2mLs of pre-warmed VCM was added to gelatin filters and 174 incubated for 10 minutes, centrifuged and tittered. 175 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 1, 2021. ; https://doi.org/10.1101/2021.05.31.21257841 doi: medRxiv preprint Method validation using SARS-C0V-2

To determine technical limits of detection of viable SARS-CoV-2 using UPAS sampling, and to correlate 177 viable particles with genomic detection, we spiked gelatin filters with 10ul of SARS-CoV-2 diluted to 10 3 , 178 10 4 , or 10 5 TCID50 units/mL over five different spots as described above (3 filters per each of the three 179 dilutions). After a brief drying period under a biosafety cabinet, filters were loaded into the UPAS and 180 run under typical conditions (16hrs at 2 LPM). The third corresponding filter for each solution was 181 processed at time of UPAS run start, to be compared with the sampled filters to determine the degree 182 that viability decreases during sampling. Filters were then dissolved into 2 mL of pre-warmed DMEM, 183 and qualitative isolation and quantitative end-point titration was determined in Vero E6 cells. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 1, 2021. ; https://doi.org/10.1101/2021.05.31.21257841 doi: medRxiv preprint 2019-nCoV_N1-P ACC CCG CAT TAC GTT TGG TGG ACC-(VIC) 6.25uM Table 1 : SARS-CoV-2 qRT-PCR primers and probes. 195

We considered the E protein concentrations as more reliable than N, because the primer probe set is 196 much more sensitive than the N. Samples with a Ct values below 36 for either gene were deemed to be 197 Hospital. The boards each determined that formal approval was not required for this study, because 212 sampling was environmental in nature, we did not propose to collect personal information such as time 213

of symptom onset or current symptomatology, and given the urgent need for this data. Moreover, as 214 personal information was not being collected, the boards felt that patient consent was not required. 215 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Furthermore, we compared recovery from samples either processed immediately after dissolving or 234 after dissolved media was placed at 4°C overnight and found no substantial difference. 235

Interestingly, SARS-CoV-2 maintained much more viability in the experiments compared to VSV, with the 236 latter undergoing a reduction of over 90% over the sampling period. As such, method development 237 experiments are required to quantify the ability of this sampling method to maintain viability for other 238 viruses of concern. 239 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 1, 2021. ; https://doi.org/10.1101/2021.05.31.21257841 doi: medRxiv preprint duct. We also sampled the cold air return that serviced all the inmate's cells and living areas twice, and 276 did not find viral RNA. 277 RNA samples were positive in 9.1% (6/66) of samples obtained with the UPAS 2.5 samplers, 13.5% (7/52) 278 with the UPAS 10 samplers, and 10.0% (2/20) samples obtained with the Coriolis samplers (Table 3) . 279

There was no significant difference in positivity rates with the different samplers (Pearson Chi-Square 280 0.59, p = 0.74). However, sampling results should not be directly compared between the UPAS and 281

Coriolis, because they were deployed for very different durations. A logistic regression was performed to 282 ascertain the effects of room and sampler type and estimated air change rate on the likelihood that a 283 positive RNA result would be observed. The logistic regression model was not statistically significant 284 ( CoV-2 RNA virus may be present at low levels in aerosols <10um in diameter, >2 m from COVID-19 294 patients in a variety of settings, but viable virus appears to be uncommon, as has been described 295 elsewhere.

[6] 296 297 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In classical terms, respiratory viruses have been considered to be spread by droplets. Large droplets 298 (e.g., > 5 microns in diameter) were believed to contaminate the immediate environment of an 299 infectious patient, including the air within 2m, leading to infection by direct deposition of virus onto 300 mucosal surfaces. In addition, large droplets settle in the proximal (within 2m) environment, leading to 301 fomite transmission where contaminated surfaces are contacted by another person prior to touching 302 their face thereby acquiring infection. In classical terms, aerosol (or "airborne") spread occurs via small 303 droplets (e.g., < 5µm) that can remain suspended in air more than 2m from a patient, leading to 304 infection of susceptible people, via inhalation at a distance. We sampled indoor air for viral RNA and viable SARS-CoV-2 virus in a large number of rooms in a variety 314 of settings and with a variety of room air change rates. We were careful to always sample two or more 315 meters from COVID-19 patients, to ensure that we were detecting virus only at distances traditionally 316 considered to be consistent with airborne transmission. Despite this variety of indoor environments, 317 viral RNA was detected infrequently. Only 10.9% of rooms contained viral RNA, and while detection 318 rates were highest in ICU rooms and rooms in long-term care facilities, the differences between room 319 types were not statistically significant. Differences in detection rates likely reflect a multiplicity of 320 factors, including each patient's infectivity and duration of illness, room size, and ventilation rates.

[24] 321 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Our mean Ct values were just over and under 34 for the N and E proteins, respectively. The Ct value was 324 under 34 for the N protein in only one room, and under 34 for the E protein in eight rooms. We did not 325 observe a significant difference of RNA positivity rates using samplers capturing larger (10μm) or smaller 326 particles (2.5μm) or non-sized particles, suggesting viral RNA may be present in a range of particle sizes. 327

Within the potential limitations of our methodology, our results suggest that aerosol transmission risk 328 beyond 2m was low in hospital rooms, at the time of our sampling. While hospitalization is more likely 329 to occur later in the disease course, when infectivity is lower, caution is still warranted. in an air handling grate, 58.3% of corridor samples, and a sample at a doorway over 2m from a patient 345 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 1, 2021. The UPAS sampler was appropriate for use in this study, and provided flexibility for collecting aerosol 364 samples in multiple environments. It is compact, lightweight and quiet, so it is easily deployable and 365 suitable for personal monitoring. In addition, its internal pump, internal battery, and lack of protruding 366 pieces or external tubing made it more acceptable for sampling in clinical environments, where clinicians 367 have limited time, and would be averse to deploying samplers that may increase contamination risks or 368 disturb patients. Its main benefit for this study was that it can be fitted with validated size-selective 369 inlets, which allowed us to collect only respiratory particles in the size fractions conventionally defined 370 as aerosols. 371

It is worth noting that aerosol sampling, and subsequent culturing, may not be a necessary indicator of 372 penitentiary prisoners many of whom were asymptomatic when diagnosed or were within a day or two 393 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Coriolis 9 0 n/a n/a n/a Long-term Care 15 2.5 Micron 2 0 n/a n/a n/a Coriolis 0 n/a n/a n/a n/a Correctional 8 2.5 Micron 4 0 n/a n/a n/a a Coriolis 0 n/a n/a n/a n/a 

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