Evidence from Whole Genome Sequencing of Aerosol Transmission of SARS-CoV-2 almost Five Hours after Hospital Room Turnover

Experimental evidence suggests that SARS-CoV-2 remains viable within aerosols with a half-life of approximately 1-3 hours, though changes in aerosol microenvironment may shorten viability to minutes. However, it remains unclear how long airborne SARS-CoV-2 can transmit infection. Whole genome sequencing of nasopharyngeal samples obtained from patients on an outbreak unit suggested in-room transmission of the delta variant, AY3 lineage, of SARS-CoV-2 to two patients admitted 1 hour, 43 minutes and 4 hours, 45 minutes after discharge of an asymptomatic infected patient. These �ndings suggest that airborne SARS-CoV-2 may transmit infection for nearly 5 hours, even in a hospital setting.


Introduction
Airborne transmission is a major source of SARS-CoV-2 infection. 2,3SARS-CoV-2 has been isolated from air samples in rooms of infected patients and in hospital locations remote from the care of patients with COVID-19, such as nurses stations on non-COVID units. 4,5In one hospital outbreak, whole genome sequencing (WGS) from air samples, staff, and patients implicated aerosol transmission as the likely mechanism of transmission. 4Experimental evidence suggests that SARS-CoV-2 remains viable within aerosols with a half-life of approximately 1-3 hours, 6,7 though changes in aerosol microenvironment may shorten viability to minutes. 8However, it remains unclear how long airborne SARS-CoV-2 can transmit infection.Here we present evidence from WGS suggesting in-room transmission of SARS-CoV-2 to two patients admitted 1 hour, 43 minutes and 4 hours, 45 minutes after discharge of an asymptomatic infected patient.

Methods
This is a molecular epidemiological analysis of a previously reported case series from a single inpatient unit at VA Boston Healthcare System (VABHS) with nosocomial transmission of the SARS-CoV-2 delta variant. 1 The outbreak occurred in July of 2021.Viral WGS was performed on nasopharyngeal swab samples, as described, 9 and used to characterize the chain of transmission between individuals. 10The IRB at VABHS deemed this work a quality improvement study and waived IRB approval.

Results
WGS identi ed the SARS-CoV-2 delta variant, AY3 lineage, and subdivided all eight individuals from the outbreak into two clusters of four (Figure).Samples from each cluster demonstrated sequence identity, and Cluster 2 differed from Cluster 1 by a single nucleotide polymorphism (SNP), the reversion mutation T29742G.In contrast, WGS on contemporary samples from two VABHS inpatients who were not on the outbreak unit and two from VA Connecticut differed from those in the outbreak clusters by 2 to 6 SNPs (Supplementary Table 1).

Cluster 1
Patient A, the presumed index case, was hospitalized for 24 days on the outbreak unit, overlapping the remaining seven persons in the outbreak clusters.Patient A was diagnosed with asymptomatic COVID-19 during a mandatory pre-discharge PCR test (Ct = 26; Outbreak Day 0).He had walked unmasked throughout the unit and eventually required a designated nursing assistant (Person B).Through contact tracing, Person B was found to be negative by antigen testing on Outbreak Day 2 but was diagnosed with asymptomatic COVID-19 on Outbreak Day 5 (Ct = 18).Patients C and D had symptomatic hospitalacquired COVID-19 on Outbreak Days 4 and 10, respectively.Both were admitted to the unit prior to the outbreak, had a negative PCR within 24 hours of admission, and converted on the unit.Contact tracing was negative in 168 masked staff member, 6 visitors, and 38 additional patients. 1Air turnover in this room was measured at 6 per hour prior to the outbreak.

Discussion
WGS may be helpful in identifying transmission chains during hospital outbreaks. 10One cluster of transmissions on this outbreak unit was from Patient A to the rest of Cluster 1 -his nursing assistant (Person B) and Patients C and D. All four persons were infected with identical versions of the SARS-CoV-2 Delta variant (AY3 lineage).Because Patient A and Patient E were hospitalized concurrently, it appears likely that Patient A also infected Patient E, and a reversion mutation (T29742G) occurred before or during this transmission event.Transmission from other sources was improbable based on extensive contact tracing and the nearly identical viral sequences identi ed in the two clusters.It is unlikely that Patient A independently infected Patients E, F and G because this would have required three identical reversion mutation events, a statistical improbability.More likely, SARS-CoV-2 in lingering aerosols or from aerosol-contaminated surfaces from Patient E remained viable for hours before infecting Patients F and G.This occurrence would imply that airborne SARS-CoV-2 is not only viable for hours in aerosols but is also capable of transmission. 6imitations of this study are its observational nature and the lack of su ciently frequent SARS-CoV-2 testing to identify the onset of infection more precisely.Likewise, the measurement of air turnover was not contemporaneous with the outbreak; therefore, we cannot be certain about the level of room ventilation during the outbreak.

Cluster 2 All
four persons in Cluster 2 spent time in the same hospital room.Patient E was hospitalized for 15 days concurrent with Patient A's hospitalization, though in a different room, and was discharged three days before Patient A's COVID-19 diagnosis.Contact tracing identi ed asymptomatic COVID-19 (Ct = 25) in Patient E eight days after discharge.Patient E was discharged at 11:00 AM on Outbreak Day − 3. Two patients without prior contact with Patient E were admitted that same day to the room previously occupied by Patient E: Patient F at 12:43 PM and Patient G at 3:45 PM.Both patients had negative COVID-19 tests four days prior to admission.Patient F was discharged one day following admission (Outbreak Day − 2) and diagnosed with asymptomatic COVID-19 (Ct = 29; Outbreak Day 1) four days after admission.Patient G was diagnosed with presymptomatic COVID-19 (Ct = 23) three days after admission, became symptomatic one day later, and transmitted SARS-CoV-2 to a visiting family member, who was diagnosed six days later with presymptomatic COVID-19 (Ct = 15; Outbreak Day 6).

Figure 1 Proposed
Figure 1