key: cord-0761859-feohcey3
authors: Lednicky, John A.; Lauzardo, Michael; Alam, Md. M.; Elbadry, Maha A.; Stephenson, Caroline J.; Gibson, Julia C.; Morris, J. Glenn
title: Isolation of SARS-CoV-2 from the air in a car driven by a COVID patient with mild illness
date: 2021-04-24
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.04.063
sha: a2218e0d11faf2066aa3ad8b4d1d92b091d7b570
doc_id: 761859
cord_uid: feohcey3

OBJECTIVE: To determine if viable virus could be isolated from the air within a car driven by a patient infected with SARS-CoV-2, and to assess the size range of the infectious particles. METHODS: We used a Sioutas personal cascade impactor sampler (PCIS) to screen for SARS-CoV-2 in a car driven by a COVID-19 patient. The patient, who had only mild illness without fever or cough and was not wearing a mask, drove the car for 15 minutes with the air conditioning turned on and windows closed. The PCIS was clipped to the sun-visor above the front passenger seat and was retrieved from the car two hours after completion of the drive. RESULTS: SARS-CoV-2 was detectable at all PCIS stages by PCR and was cultured from the section of the sampler collecting particles in the 0.25 to 0.50 μm size range. CONCLUSIONS: Our data highlight the potential risk of SARS-CoV-2 transmission by minimally symptomatic persons in the closed space inside of a car and suggest that a substantial component of that risk is via aerosolized virus.

While we have learned a great deal about transmission of SARS-CoV-2 since the beginning of the current pandemic, questions remain about the exposure risk in different settings, and the contribution of various modes of transmission to virus spread Somsen et al., 2020; WHO, 2020; CDC, 2020a) . In particular, there is continuing uncertainty about the relative contribution of larger virus-laden respiratory particles at close distances, compared with virions in aerosols at close or longer ranges, to spread of the virus. There are now multiple epidemiology studies consistent with aerosol spread of SARS-CoV-2 within closed spaces (Park et al., 2020; Hamner et al., 2020) and the virus has been shown to remain infective in laboratorygenerated aerosols for at least 16 hours Fears et al., 2020) . Data from our group and others have J o u r n a l P r e -p r o o f documented the presence of the virus in aerosols in patient settings by RT-PCR (Rahmani et al., 2020; Lednicky et al., 2020a; Santarpia et al., 2020) . However, molecular detection of SARS-CoV-2 RNA does not necessarily correlate with risk of developing COVID-19, since only viable virions can cause disease. Subsequently, we have reported isolation of viable SARS-CoV-2 from the air within the room of hospitalized COVID-19 patients (Lednicky et al., 2020b) .

The current study was undertaken to assess SARS-CoV-2 transmission in a "real life" setting, outside of a medical facility. In epidemiologic studies, public transportation vehicles have been identified as a risk factor for transmission by CDC (CDC, 2020b), and studies from Singapore have reported an odds ratio for infection of 3.07 [95% CI 1.55-6.08] for persons sharing a vehicle with an infected person (Ng et al., 2020) . In this setting, we were interested in documenting that viable virus could be isolated from the air of a car being driven by a person infected with SARS-CoV-2. We were also interested in determining the size distribution of airborne respiratory secretions that contain virions to provide some guide to the contribution of droplets and aerosols to transmission risk. To address these questions, we asked a COVID patient with minimal symptoms to drive her car for a short period of time with a Sioutas personal cascade impactor sampler (PCIS) (Misra et al., 2002; Lednicky et al., 2013) clipped onto the sunvisor above the passenger seat next to her, to permit collection of air samples for screening for the virus.

The Sioutas Personal Cascade impactor sampler (PCIS) separates airborne particles in a cascading fashion and simultaneously collects the size-fractionated particles by impaction on polytetrafluoroethylene (PTFE) filters) (Misra et al., 2002; Lednicky et al., 2013) . It has J o u r n a l P r e -p r o o f collection filters on four impaction stages (A-D), and an optional after-filter can be added onto a 5th stage (E). The PCIS separates and collects airborne particulate matter above the cut-point of five size ranges: >2.5 μm (Stage A), 1.0 to 2.5 μm (Stage B), 0.50 to 1.0 μm (Stage C), 0.25 to 0.50 μm (Stage D), and <0.25 μm (collected on an after-filter) (Figure 1 ). PTFE filters (Teflon filters) can collect particles at high efficiency above the cut-points without the need for coatings (Misra et al., 2002) , which is advantageous as various coatings reduce the recovery efficiency of viable virus. For the collection of airborne viruses, the filters are not prewetted with methanol prior to use, which helps preserve virus viability (Fabian et al., 2009 ). For the current study, a PCIS (SKC, Inc., catalog number 225-370) unit was used with a Leland Legacy pump (SKC, Inc., cat number 100-3002) and operated at a flow rate of 9 L/min. PTFE filters (25 mm, 0.5 μm pore, SKC, Inc. catalog number 225-2708) were used for the collection stages A to D, and a PTFE after-filter (37 mm, 2.0 μm pore, SKC Inc., catalog number 225-1709) for stage E. The pump's operating flow rate (9 L/min) was calibrated by measuring volume displacement using a Defender Primary Standard Calibrator (SKC, Inc., catalog number 717-510H).

Within 30 minutes of the termination of air-sampling, the PCIS filters were individually immersed in 1 ml of recovery solution (PBS with 0.5% w/v BSA fraction V and 0.2 M surcose) 11 for 30 minutes at room temperature to help rehydrate and dislodge virions stuck on the filter surfaces. The filters and fluid were then transferred to a sterile plastic petri dish, and the filters scraped with flocked swabs pre-wetted with recovery solution and residual fluid in each swab extruded into the liquid corresponding to each filter. The recovery solutions were concentrated by centrifugation in Amicon Ultra-15 centrifugal filter units with Ultracel-100 membranes with a molecular mass cutoff of 100 kDa (Millipore, Bedford, MA) at 4,000 × g for 12 min to a volume J o u r n a l P r e -p r o o f of approximately < 400 μL, and the concentrates adjusted to 400 µL by addition of recovery solution. They were then aseptically transferred to sterile plastic cryotubes with O-ring seals, and the tubes stored in a locked -80°C freezer for subsequent analyses.

After one thaw on ice, RNA was extracted from 140 µl of material extruded off the PCIS filters using a QIAamp Viral RNA Mini Kit and the purified bound RNA eluted from the RNA-binding silicon columns in a volume of 80 µl. rRT-PCR was subsequently used to detect SARS-CoV-2 RNA and was performed using primer system Led-N-F, Led-N-R, and Led-N-probe. 11 Briefly, vRNA was denatured for 5 min at 67°C in the presence of SUPERase-In RNase inhibitor (Invitrogen Corp.), cooled rapidly, and 25 µl rtRT-PCR performed in a BioRad CFX96 Touch Real-Time PCR Detection System using 5 μL of purified vRNA using the following parameters: 400 nM final concentration of forward and reverse primers and 100 nM final concentration of probe using SuperScript TM III One-Step RT-PCR system with Platinum TM Taq DNA Polymerase (Thermo Fisher Scientific). Cycling conditions were 20 minutes at 50 °C for reverse transcription step, followed by 2 minutes at 95 °C for Taq polymerase activation step, then 44 cycles of 15 seconds at 95 °C of denaturing, 30 seconds at 57 °C for annealing, and 20 seconds at 68 °C for extension (Lednicky et al., 2020b) . The number of viral genome equivalents present in each sample was estimated from the measured quantification cycle (Cq) values, and was attained by using 10-fold dilutions of calibrated plasmids containing an insert of the SARS-CoV-2 Ngene as previously described (Lednicky et al., 2020b) .

Attempts to isolate SARS-CoV-2 were performed in a BSL3 laboratory by trained analysts wearing full head-covering powered air purifying respirators and appropriate PPE. After one thaw on ice, 150 μL aliquots of PCIS fluids were inoculated onto newly confluent Vero E6 cells, which were then incubated at 37 °C in a humidified 5% CO2 incubator. Mock infected cells were J o u r n a l P r e -p r o o f maintained in parallel. The cells were re-fed with cell growth medium containing reduced serum (3% fetal bovine serum) every three days (Lednicky et al., 2020b) . Sanger sequencing was performed on RNA extracted from the cell growth medium. As a secondary check, NGS was also performed using an Illumina MiSeq platform, as previously described (Lednicky et al., 2020b) . The sequencing reactions were performed at different locations by different personnel.

Verbal consent for sample collection was obtained from the driver of the car. The work was reviewed and approved by the University of Florida IRB.

The patient, who was in her twenties, had initially presented to clinic with a one-day history of mild fatigue, nasal congestion, and sore throat, following exposure to a roommate who had a laboratory-confirmed diagnosis of COVID-19. The patient denied fever, cough, shortness of breath, or other symptoms, and had a normal physical exam. In testing performed by the UFHealth clinical laboratory, a nasopharyngeal swab collected at the time of presentation was positive by RT-PCR. The patient was advised to isolate at home for a 10-day period of time, and appropriate contact tracing was initiated.

Two days after the diagnostic sample was obtained, the patient agreed to have the PCIS placed in her car (an older model Honda Accord) for the drive from the clinic to her home. Her symptoms at that time were minimal, with no cough. The PCIS was attached to the sun-visor on the passenger side of the car, approximately 3 feet from the patient's face and with the intake port pointing toward the roof of the car, with the pump assembly placed on the front passenger seat.

During the 15-minute drive the patient was not wearing a mask. The air conditioner in the car J o u r n a l P r e -p r o o f was on and windows were closed: during the drive the temperature within the car's cabin ranged from 24.2 to 22.8 °C and relative humidity fluctuated from 42.5% to 55.2%; outside temperature was 32 °C and relative humidity 99%.

Two hours after the patient's arrival home, an investigator wearing personal protective equipment (N95 mask, gloves, and a Tyvek laboratory coat) opened the car and turned off the pump of the air sampler, and transferred the PCIS-pump assembly into a sealed container and decontaminated the outer surface of the container. A total collection time of 135 min was thus used to sample approximately 1.22 m 3 of air within the vehicle.

SARS-CoV-2 RNA was detected on filters A to D, suggesting that the PCIS had collected a range of particle sizes containing SARS-CoV-2 virions or other material (possibly cell debris) containing SARS-CoV-2 RNA ( Table 1) . More of the SARS-CoV-2 RNA material was collected on filter D than that on filters A-C and E combined. Sanger sequencing was performed on RNA extracted from the cell growth medium corresponding to PCID filter D (Table 2) and suggest that a substantial component of that risk is via aerosolized virus.

Internal funding was provided by the University of Florida Emerging Pathogens Institute. Study sponsors had no role in study design, collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.

the study was reviewed and approved by the University of Florida Institutional Review Board. 

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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.

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.J o u r n a l P r e -p r o o f