During the coronavirus infectious disease 19 (COVID-19) pandemic, droplet transmission has been considered the most significant transmission route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although other routes such as aerosol, fecal-oral, and indirect transmission via fomites may contribute to the rapid global dissemination of the virus. The relative importance of aerosols versus droplets in the transmission of respiratory infections is difficult to distinguish, since particles of both aerosol and droplet size are generated for example when talking1,2. Aerosols are smaller than droplets and thus remain airborne longer, enabling transmission at greater distances and over longer periods of time.
In recent studies, extensive environmental contamination of SARS-CoV-2 in hospital settings has been demonstrated, and viral RNA has been found both in air samples and in samples from air vent openings in isolation rooms3,4. In this study from a COVID-19 hospital ward, we detected SARS-CoV-2 RNA in and near air vent openings in isolation rooms and in filters and fluid sample collections in the ventilation system at the top of the hospital building. Our findings indicate both aerosol dispersion of SARS-CoV-2 and possible long-distance dissemination of SARS-CoV-2 via ventilation air flow.
Surfaces were swabbed using sterile nylon flocked swabs (Copan eSwab, Copan Italia SpA, Italy) moistened in sterile viral transport medium, containing Hank’s balanced salt solution (Gibco, UK) supplemented with 2% fetal bovine serum (Gibco, USA), 100µg/ml Gentamicin, and 0.5 µg/ml Amphotericin B5. Round ceiling vent openings were swabbed around the inside of the entire opening (circumference ca 25 cm). Swabs were placed in transport medium, sealed and stored at +4 C until analysis. Collections were performed on April 17 and 28, 2020. Indoor relative air humidity was 30-31 % and temperature 20-21°C.
Exit ventilation from each COVID-19 ward (Figure) leads to separate HEPA filter systems, distance measured to between 49 and 56 meters (Table). Adjacent inspection hatches upstream from the HEPA filters were opened, and internal 30 x 30 cm surfaces swabbed as described. One (of six) 60 x 60 cm laminate F7 HEPA filter sections was removed from each system and three filter samples (3 x 3 cm) were randomly cut out using sterilized scissors, placed in vials containing 2,5 ml of transport medium, and stored at +4°C until analysis. The removed filters had been routinely replaced one month prior to collection.
Fluid sample collection was performed by placing open petri dishes with cell medium (Dulbecco’s Modified Eagle’s medium; DMEM, (Gibco, Code: 13345364)), suspended 15 cm below ceiling vent openings (in ward rooms) for 24 h, or placed within central vent ducts via inspection hatches, for 3 h. Medium exposed to air in 19 ward rooms were combined to three pools before being applied to Vero E6 cells and subjected to rRT-PCR.
RNA was extracted using QIAamp viral RNA kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. SARS-CoV-2 envelope small membrane protein € and nucleocapsid (N) were amplified by rRT-PCR using the SuperScript III OneStep rRT-PCR System with Platinum® Taq DNA Polymerase kit (Invitrogen, Cat. No. 12574026)6–8.
Vero E6 cells (green monkey kidney cells (ATCC® CRL-1586™)) grown in T25 flasks and 6 well plates were used for infection, and cytopathic effect (CPE) was observed daily. Increase in viral load was determined using rRT-PCR, 96 hours post inoculation for T25 flasks and every 24 hours for up to 96 hours post infection (hpi) in 6-well plates. Supernatant was applied to uninfected cells 96 hpi, twice, resulting in three passages.
In two consecutive sampling rounds, both SARS-CoV-2 N and E gene RNA were detected in seven out of 19 vent openings, while 11 days later, four vents were positive for both genes. Cycle threshold (Ct) values varied between 35.31 and 39.78 (Table). All three pooled cell medium samples from patient room ceilings were positive for both genes; Ct values ranged between 33.41 and 36.64.
At attic level (Figure), samples extracted from the main filters serving each ward were predominantly positive for both genes. One of three exposed open petri dishes with cell medium were positive for both genes. Swabs taken from internal surfaces of three central ventilation channels were all negative (Table).
No significant CPE nor decrease in rRT-PCR Ct values were seen after three passages on Vero E6 cells from samples retrieved from ward vent openings or central ventilation ducts or filters. From the pooled samples collected directly in cell medium in patient rooms, SARS-CoV-2 RNA was detected in three subsequent passages with slightly decreasing Ct values, despite dilution in each passage. We could not, however, observe any significant CPE in this experiment.
Several aspects during the COVID-19 pandemic support the risk of airborne transmission of SARS-CoV-2. First, mounting evidence for pre- and asymptomatic transmission, where the spread of droplets through coughing and sneezing cannot be a major factor, must raise questions about aerosol transmission9. Second, aerosols generated by speech could theoretically contain enough SARS-CoV-2 virus particles to support transmission, and these aerosols can remain airborne for up to ten minutes10. In addition, coronaviruses can be emitted in aerosols through normal breathing11. Third, field studies in hospital wards have detected SARS-CoV-2 RNA both in vent openings and in the air3,4. These findings are not unexpected seeing as similar observations have been made for both SARS and Middle East Respiratory Syndrome (MERS) 12–14.
In this study, we found SARS-CoV-2 RNA in vent openings in ward rooms harboring COVID-19 patients. Viral RNA was also detected in fluid placed in open dishes suspended below vent openings. Similar levels of viral RNA were detected in exhaust filters and open petri dishes with cell medium at least 44 to 56 meters from the COVID-19 wards. Only a small fraction of each filter was analyzed implying that the large number of particles emanating from COVID-19 wards can disperse to greater distances than can be explained by droplet transmission routes. In previous studies, the effect of ventilation has not shown any obvious impact on the risk for spread of droplet-transmitted diseases, probably since droplets are more governed by gravity15. Furthermore, the ventilation system in the investigated hospital building has a relatively low air flow; between 1,7 and 3 total air changes per hour (ACH) for each room, depending on room volumes. The recommendation for airborne isolation rooms is 12 ACH in most guidelines15.
Ongoing oxygenation therapies, such as High Flow Nasal Cannula (HFNC) oxygenation, in each room did not apparently correlate to detection of SARS-CoV-2 RNA in vent openings. This raises the question if the risk for aerosol transmission should be considered in more situations than during potentially generating procedures such as HFNC, which is further corroborated by the studies on aerosols generated when speaking and breathing10,11. Results differed in ward rooms between sampling occasions, which could be due to varying disease progression for the occupying patients. At two sampling occasions, vent openings were positive for both N and E genes despite the rooms having been evacuated and routinely cleaned. This suggests that detection also could result from viral shedding by previous patients and calls for further studies on how long SARS-CoV-2 RNA can be detected in the environment, with the accompanying risk for transmission via fomites.
In this study we could not demonstrate infectious capability of the virus, when inoculated on Vero E6 cells, from samples in either vent openings, exhaust filters or by collection in cell medium. This is likely due to the pathogens rapidly drying in the vents or inadequate amounts of virus collected near vent openings or in front of exhaust filters. Furthermore, admitted patients in the ward were between day 5 and 23 after symptom onset (Table). There is accumulating evidence that COVID-19 contagiousness peaks shortly prior to symptom onset16. This implies that the patients in this study may be in a less contagious phase of COVID-19 disease, which is consistent with the findings that SARS-CoV-2 infectivity appears to be low eight days after symptom onset 17,18. Nevertheless, during dispersal from a patient to ventilation, and over considerable distances, the virus may still retain infective capability. This should be even more important concerning patients in earlier phases of disease. The presented findings must be regarded as indicative of possible aerosol dissemination of SARS-CoV-2, especially considering the distance SARS-CoV-2 RNA was dispersed. We propose this should raise serious concerns of potential transmission through the airborne route.