The size and culturability of patient-generated SARS-CoV-2 aerosol

The study addresses whether patients with COVID-19 generate infectious aerosols across particle sizes that can facilitate airborne transmission. Early in the pandemic, SARS-CoV-2 was considered primarily a droplet/contact-spread pathogen, but accumulating evidence supports aerosol transmission. Debate exists over the 5 µm cutoff traditionally used to define airborne transmission, with recent work arguing for a broader continuum of aerosol sizes. To establish airborne transmission, it must be shown that ill individuals produce infectious aerosol small enough to be inhaled, that such aerosol remain viable long enough to expose others, and that inhalation can lead to infection. Prior studies have cultured influenza virus from particles <5 µm, have cultured SARS-CoV-2 from air near infected individuals, demonstrated multi-hour aerosol stability for SARS-CoV-2 in the absence of sunlight, widespread environmental RNA contamination, and outbreak investigations suggestive of airborne spread. Given ACE2 expression throughout the respiratory tract, inhalation is a plausible route. Motivated by calls from the National Academies to clarify aerosol transmission of SARS-CoV-2, this study examines the presence and infectivity of SARS-CoV-2 across aerosol size fractions produced by hospitalized patients, including those generated during normal respiration, vocalization, and coughing.

The paper situates its research within several lines of evidence: (1) previous demonstrations that viable influenza can be detected in exhaled particles <5 µm; (2) reports of cultured, viable SARS-CoV-2 in air samples collected near infected individuals; (3) laboratory evidence showing SARS-CoV-2 retains infectivity in aerosols for hours (attenuated by simulated sunlight); (4) extensive detection of SARS-CoV-2 RNA in air and on surfaces in patient care areas; (5) outbreak investigations (e.g., choir practice, restaurant) consistent with airborne transmission; and (6) biological plausibility via ACE2 expression throughout the respiratory tract. The authors also discuss the inadequacy of a rigid 5 µm cutoff for distinguishing droplet versus aerosol transmission, advocating a spectrum of sizes contributing to airborne spread. These works collectively motivate direct measurement and culturability testing of patient-generated aerosols by size fraction.

Design and setting: Aerosol sampling was conducted around six COVID-19-positive patients across five rooms in two hospital wards (Ward 5 and Ward 7) on three separate days in April 2020. Ward 7 rooms operated under negative pressure to hallways; Ward 5 had the entire ward under negative airflow. Ventilation configurations differed between wards; room surfaces were disinfected daily. Aerosol measurement and collection: An Aerodynamic Particle Sizer Spectrometer (APS 3321, TSI) measured aerosol concentrations and size distributions (0.5–20 µm) at 1-minute intervals for 30 minutes per room (10 minutes for one room due to power loss). A collocated NIOSH BC251 sampler, operated at 3.5 L/min, size-segregated aerosol into three fractions: >4.1 µm (15 mL conical), 1–4 µm (1.5 mL conical), and <1 µm captured on a 37 mm gelatin filter (Sartorius) to preserve viral integrity. Samplers were placed at the foot of the patient’s bed (~1.1 m above floor). A blank gelatin filter served as a daily handling control. Sample recovery and RNA detection: Within 1–2 hours of collection, samples were eluted in PBS (5 mL for >4.1 µm, 1 mL for 1–4 µm, 10 mL for <1 µm by dissolving the gelatin filter). RNA was extracted from 400 µL using Qiagen DSP Virus Spin Kit with negative extraction controls. rRT-PCR targeted the SARS-CoV-2 E gene (WHO protocol) using Invitrogen Superscript III One-Step system on a QuantStudio 3, with positive synthetic DNA and no-template controls; each sample was run in triplicate. A standard curve was built from RNA extracted from cultured SARS-CoV-2 (BEI USA-WA1/2020) spanning five logs (starting at 1×10² pfu/mL by plaque assay). The exponential fit Equivalent Viral Titer (pfu/mL) = 6.0E9 × e^(−0.707×Ct) was used to convert Ct to equivalent pfu (e-pfu), and an empirically determined ratio of 1.35×10⁶ ± 6.29×10⁵ RNA copies per pfu was applied to estimate RNA copies per liter of air. Cell culture and replication assessment: Vero E6 cells in supplemented DMEM (10% heat-inactivated FBS, penicillin/streptomycin, amphotericin B) were inoculated with 100 µL of undiluted aerosol sample. A mock infection control used 100 µL of extracted SARS-CoV-2 RNA (1×10² TCID50/mL). Additional infection controls used stock virus adjusted to supernatant concentrations of approximately 1.6, 1.6×10¹, and 1.6×10² pfu/mL. Cultures were maintained at 37 °C, 5% CO₂. Supernatant was sampled on day 1 and day 5 or 6 for RNA extraction and rRT-PCR; percent change from day 1 to final day was calculated. Significant increases (Student’s t test, P < 0.05) indicated replication. After 6 days, all supernatants and lysates were harvested. Protein and virion detection: For samples with >90% confidence of replication, western blots probed for SARS-CoV-2 nucleocapsid (N) protein (40–55 kDa band) with GAPDH as loading control. Transmission electron microscopy (TEM) was performed on fixed, processed sections using a Tecnai G2 Spirit TWIN at 80 kV to visualize virions. Aerodynamic data analysis: APS dM/dlogD mass distributions (assuming unit density) were averaged across 1-minute measurements (excluding the <0.523 µm bin). Two lognormal modes were fit to each room’s average distribution using Excel Solver minimizing least squares to estimate mode mass (M), mass median aerodynamic diameter (dₒg), and geometric standard deviation (σg). Mode masses were integrated over 0.542–0.97 µm, 1.04–3.79 µm, and 4.07–9.85 µm to correspond to BC251 stages, and normalized for comparison with RNA concentrations from BC251 fractions.

• SARS-CoV-2 RNA was detected by rRT-PCR in all three BC251 size fractions for all six rooms (18/18 aerosol samples positive for RNA).
• Evidence of replication in culture: 3 of 18 samples (all from the <1 µm fraction) showed statistically significant increases in RNA after 5–6 days in Vero E6 culture (P < 0.05), specifically rooms 7B, 5A, and 5C. Two samples from the 1–4 µm fraction (7B and 5C) showed increases with P values between 0.05 and 0.1 (suggestive but not definitive). No samples from the >4.1 µm fraction showed significant replication; one showed a decline in RNA (growth ratio <1).
• Controls: Mock infection with extracted RNA became undetectable by rRT-PCR after 1 day, indicating observed increases reflect replicating virus rather than free RNA. Replication was observed in culture controls at approximately 1.6×10¹ and 1.6×10² pfu/mL, but not at ~1.6 pfu/mL.
• Protein and virion detection: Western blot detected SARS-CoV-2 N protein in all but one of the samples with >90% replication confidence (negative in room 7B <1 µm). TEM visualized intact SARS-CoV-2 virions in the submicron sample from room 5C.
• Aerosol size distributions: APS data showed two lognormal modes. The small mode had mean diameters 0.64–0.80 µm with σg 1.17–1.30 across rooms; large mode varied more. A statistically defensible correlation between APS modes and RNA in BC251 fractions could not be established, likely due to background aerosol contributions.
• Overall, infectious SARS-CoV-2 was most confidently associated with submicron aerosol (<1 µm), with possible but less certain presence in the 1–4 µm range, and no culture-positive detections in >4.1 µm samples under these sampling conditions.

Findings demonstrate that hospitalized COVID-19 patients generate fine aerosols containing replication-competent SARS-CoV-2, particularly in the submicron range. These particle sizes align with those produced during normal breathing, vocalization, and coughing, with origins in the bronchiolar and laryngeal regions. The absence of culture-positive detections in the >4.1 µm fraction may reflect true lower infectivity in larger droplets or methodological limitations: the BC251’s inertial impaction in dry conical tubes could degrade virions in larger stages, whereas the gelatin filter in the <1 µm stage may better preserve infectivity. Thus, while infectious virus was clearly present <1 µm and possibly up to 4 µm, the study cannot exclude infectious virus in larger particles. High RNA-to-pfu ratios observed (potentially reflecting defective interfering particles) underscore that RNA detection overestimates infectious virion abundance, both in culture and clinical samples. Together with prior evidence of aerosol stability and epidemiologic data, these results support airborne transmission potential and the need for efficient respiratory protection and airborne isolation precautions in healthcare and community settings.

SARS-CoV-2 RNA is present in respired aerosols <5 µm that are generated during routine respiratory activities, and a subset of these RNA-containing aerosols—particularly in the submicron range—harbor intact, replication-competent virus. These findings support a role for fine aerosols in SARS-CoV-2 transmission and reinforce implementing effective airborne infection prevention measures, including efficient respiratory protection, in healthcare and public settings. Given the likely importance of airborne transmission, aerosol prevention strategies should be emphasized, especially in crowded environments. Future research should refine aerosol sampling methods to preserve viral infectivity across sizes, disentangle patient-generated aerosols from background, and quantify infectious dose across particle sizes and activities.

• Sampling and preservation: The BC251 sampler’s first two stages use dry inertial impaction, which may degrade viral integrity, potentially underestimating infectious virus in 1–4 µm and >4.1 µm fractions. Gelatin filter use in the submicron stage may have preferentially preserved infectivity there.
• Background aerosol confounding: APS size distributions could not be separated into patient-generated versus background aerosol without extensive background characterization, limiting source attribution.
• Small sample size and setting: Only six patients across five rooms were studied, all in hospital settings, limiting generalizability.
• Culture and detection sensitivity: High RNA-to-pfu ratios and potential defective interfering particles complicate interpretation of RNA quantities versus infectious virus. TEM confirmed virions in only one sample, and it remains unclear whether observed virions were produced during culture.
• Temporal snapshot: Single 30-minute sampling periods per room may not capture temporal variability in aerosol generation and infectivity.