Live SARS‐CoV‐2 is difficult to detect in patient aerosols

As the COVID19 pandemic rages, there has been much debate regarding the importance of bioaerosols in SARSCoV2 transmission. Circumstantial evidence indicates that aerosol transmission is a likely contributor to the current pandemic,13 yet research teams have had difficulty isolating live virus when using traditional aerosol sampling techniques.46 To our knowledge, thus far only two research teams have successfully cultured airborne SARSCoV2 outside of laboratory simulations,7 with one team finding evidence of viral replication in the absence of cytopathic effect.8,9 Despite the demonstrated challenges in capturing suspended live virus, respiratory transmission is now thought of as the primary mode of SARSCoV2 infection. By contrast, SARSCoV2 has been readily cultured from nasopharyngeal (NP) swabs, saliva, blood, stool, and semen.4,1012 Mounting evidence indicates that COVID19 patients are most infectious within the first eight days following symptom onset,4,12,13 with some outliers shedding live virus for up to 18 days.13,14 Increased viral load is associated with better odds of culturing virus, with cutoff values reported at 24 and 34 RTPCR cycle thresholds.4,13 Building on previous work carried out by our team,15 we sought to enroll home isolated SARSCoV2 positive patients early in their disease progression to estimate the viability of the virus in biological, environmental, and bioaerosol samples and assess aerosol transmission of SARSCoV2. From October 2020 to January 2021, we visited eight patients in their homes in and around Durham, North Carolina soon after they were confirmed by molecular assay to be infected with SARSCoV2. After informed consent was obtained, we asked patients to complete a brief questionnaire, and to permit the collection of a NP swab, passive saliva sample, fomite swabs, and bioaerosol samples. Patients were also asked to selfcollect a rectal swab sample. All study procedures were approved by the Duke University Institutional Review Board (Pro00105055). Bioaerosol sampling was carried out using National Institute for Occupational Safety and Health (NIOSH) BC 251 aerosol samplers, placed ~1.5 meters off the ground at distances of ~1 meter, 1.4 meters, 2.2 meters, and 3.2 meters from the participant's head. The participants were asked to remain stationary in their room and the samplers were run for approximately two hours at a calibrated flowrate of 3.5 Lair/min. 15 SKC 20ml BioSamplers (SKC, Inc., Pennsylvania, USA), prepared with 16 mL phosphatebuffered saline (PBS) and 0.5% bovine serum albumen (BSA), were placed beneath the NIOSH samplers on one side of the room and run simultaneously at the recommended flow rate of 12.5 Lair/min. These samplers are designed to capture viral matter in liquid media to enhance viability. It is important to note that we have previously used both types of samplers to capture live influenza A virus.1619


| DE AR ED ITOR COWLING
As the COVID-19 pandemic rages, there has been much debate regarding the importance of bioaerosols in SARS-CoV-2 transmission.
Circumstantial evidence indicates that aerosol transmission is a likely contributor to the current pandemic, 1-3 yet research teams have had difficulty isolating live virus when using traditional aerosol sampling techniques. [4][5][6] To our knowledge, thus far only two research teams have successfully cultured airborne SARS-CoV-2 outside of laboratory simulations, 7 with one team finding evidence of viral replication in the absence of cytopathic effect. 8,9 Despite the demonstrated challenges in capturing suspended live virus, respiratory transmission is now thought of as the primary mode of SARS-CoV-2 infection. By contrast, SARS-CoV-2 has been readily cultured from nasopharyngeal (NP) swabs, saliva, blood, stool, and semen. 4,[10][11][12] Mounting evidence indicates that  patients are most infectious within the first eight days following symptom onset, 4,12,13 with some outliers shedding live virus for up to 18 days. 13,14 Increased viral load is associated with better odds of culturing virus, with cut-off values reported at 24 and 34 RT-PCR cycle thresholds. 4,13 Building on previous work carried out by our team, 15 we sought to enroll home isolated SARS-CoV-2 positive patients early in their disease progression to estimate the viability of the virus in biological, environmental, and bioaerosol samples and assess aerosol transmission of SARS-CoV-2.
From October 2020 to January 2021, we visited eight patients in their homes in and around Durham, North Carolina soon after they were confirmed by molecular assay to be infected with SARS-CoV-2.
After informed consent was obtained, we asked patients to complete a brief questionnaire, and to permit the collection of a NP swab, passive saliva sample, fomite swabs, and bioaerosol samples. Patients were also asked to self-collect a rectal swab sample. All study procedures were approved by the Duke University Institutional Review Board (Pro00105055).
Bioaerosol sampling was carried out using National Institute for Occupational Safety and Health (NIOSH) BC 251 aerosol samplers, placed ~1.5 meters off the ground at distances of ~1 meter, 1.4 meters, 2.2 meters, and 3.2 meters from the participant's head.
The participants were asked to remain stationary in their room and the samplers were run for approximately two hours at a calibrated flow-rate of 3.5 L air /min. 15 SKC 20-ml BioSamplers (SKC, Inc., Pennsylvania, USA), prepared with 16 mL phosphate-buffered saline (PBS) and 0.5% bovine serum albumen (BSA), were placed beneath the NIOSH samplers on one side of the room and run simultaneously at the recommended flow rate of 12.5 L air /min. These samplers are designed to capture viral matter in liquid media to enhance viability.
It is important to note that we have previously used both types of samplers to capture live influenza A virus. [16][17][18][19] Biological samples and fomite swabs were collected and processed as previously described, 15  Cells and supernatant were harvested 7 days post-inoculation. RNA extracts were then screened for SARS-CoV-2 with the real-time RT-PCR assay and considered positive when the CT value was at least 2 points below the original result and CPE was present. 15 All participants presented with mild to moderate illness. The majority of the participants were females (n = 7, 87.5%). The predominantly represented race was white (n = 5, 62.5%), with two Black participants (25.0%), and one Asian (12.5%). One participant identified as Hispanic, and a second as Indian. The mean age was 41.4 years, with a range of 29 to 53 years. Among all participants, five lived in a private house, one in an apartment complex, and two in a residential shelter.
Participants were typically enrolled within three days of symptom onset (one patient was enrolled on day 8), and reported experiencing between zero and seven common COVID-19 symptoms at time of enrollment, with cough, fatigue, and body ache the most frequently listed (Table S1)

E TH I C S A PPROVA L
All study procedures were approved by the Duke University Institutional Review Board (Pro00105055).

ACK N OWLED G EM ENTS
We thank Andrew Pekosz, PhD, Professor and Vice Chair of the

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/irv.12860.

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.
Fu n ding info r mat io n This study was supported by Professor Gray's discretionary funding and grants from Duke University's Clinical Translational Science Institute and Duke Medicine and Engineering (MEDx).

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/irv.12860.

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.