Several aspects during the COVID-19 pandemic support the risk of aerosol 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 transmission31. 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 minutes20. In addition, coronaviruses can be emitted in aerosols through normal breathing32. Third, field studies in hospital wards have detected SARS-CoV-2 RNA both in vent openings and in the air14–17. These findings are not unexpected seeing as similar observations have been made for both SARS and Middle East Respiratory Syndrome (MERS) 8,33,34.
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 three investigated COVID-19 wards. Only a small fraction of each filter was analyzed implying that a 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 gravity35. 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 infection isolation rooms is 12 ACH in most guidelines35. Notably, the relative air humidity in the investigated environment was low, between 30 and 31%. Low air humidity has recently been suggested to increase the risk of airborne SARS-CoV-2 dispersal36,37.
We initiated this study by performing rRT-PCR on numerous surface and filter samples. Detection of SARS-CoV-2 as well as other coronavirus RNA in ventilation openings has been reported before.10,15,38 However, the detection of viral RNA in the exhaust filters over 50 meters from patient care areas was unexpected. In response to these findings, we found it vital to rapidly address the question of infective ability in order to determine the immediate risk of infection for uninfected patients, personnel working in the investigated wards and service personnel that might be exposed while working with the ventilation systems. We therefore employed the ad hoc methods described above in an attempt to determine the infective ability of the samples. We are aware that there are several potential limitations to the employed sampling methods in fluid traps; the likelihood of viral particles being deposited in fluids by gravity, the length of time the viral particles retain infective ability, concentration and increased osmolarity of the cell medium by evaporation as well as pH increase due to oxygen exposure during sampling. We have not determined whether the detected RNA could be from viral particles that have been inactivated by antibodies, seeing as a majority of the patients admitted to at least one of the wards were in later phases of COVID-19 disease at both collection dates (Table 2), and may have likely developed an immune response. Even though we could not determine infective capability of virus collected in cell medium, we repeatedly detected SARS-CoV-2 RNA using this method. The placement of the petri dishes, either just below the ceiling in ward rooms or at distances around at least 50 meters from patients in central vent ducts indicates that dispersal by means other than larger droplets must occur, since larger droplets are considered to precipitate by gravity within one or two meters from a source5. Although RNA could be detected in samples from ward rooms and central ventilation ducts, no infectivity was seen after inoculating samples on susceptible cells. This collection method was adopted in order to rapidly address the question as to what threat the RNA findings may infer in a clinical setting. Several explanations for these results may be identified. First, the Ct values are close to the detection limit, indicating that there were few viral copies in theses samples. Also, many of the admitted patients at this time point (later than other samplings in this study) were in late phases of COVID-19 or cleared of infection. We chose to report this as we could detect SARS-CoV-2 RNA in these samples, and that droplets do not appear to be a plausible explanation for these findings as droplets could unlikely follow a ballistic pathway from patient into the petri dished at 2.5 m height, and in all three pools. It is important to continue to develop effective sampling methods in order to determine infective ability of SARS-CoV-2 as well as differentiating between patients in early and late phases of disease. Since we are aware of these technical limitations, we have recommended service personnel to take adequate protective measures while working with the ventilation systems as we cannot definitively repudiate the risk of infection from contaminated air.
Ongoing oxygenation therapies, such as High Flow Nasal Cannula (HFNC) oxygenation, in each room did not apparently correlate to detection, or Ct values, of SARS-CoV-2 RNA in vent openings (Table 2). This raises the question if the risk for airborne transmission should be considered in more situations than during potentially aerosol generating procedures such as HFNC6. This is further corroborated by the studies on aerosols generated when speaking and breathing20,32. Results differed in ward rooms between the two samplings of vent openings, which could be due to varying disease progression for the occupying patients. Some vent openings were positive for both N and E genes despite the rooms having been evacuated and routinely cleaned (Table 2). 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. Alternatively, detection of viral RNA in the ventilation systems could arise from such activities as handling bed linens or cleaning which may disturb viral particles from textiles or surfaces and displace them into the air, and that these virions have dried and been rendered inactive. On the other hand, RNA deterioration after inactivation could limit the extent of this source of RNA found in HVAC systems.
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 directly 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. Also, collection directly in cell medium does not appear to have been performed previously and these results should be interpreted cautiously. Furthermore, admitted patients in the ward were between day 5 and 23 after symptom onset (Table 2). There is accumulating evidence that COVID-19 contagiousness peaks shortly prior to symptom onset2,31,39. 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 39,40. Nevertheless, during dispersal from a patient to ventilation, and over considerable distances, the virus may still retain infective capability. RNA was also detected in containers placed at ceiling level, demonstrating that viral particles were airborne during these specific periods, at not only deposited on fomites over longer, uncertain duration. We speculate that the risk of infection by exposure to ventilation system air is presumably very low, due to dilution of viral load and drying. Nevertheless, the apparent capability of the virus to be transported in air, as we present here, should raise concerns for the risk of infection in smaller, confined spaces in close proximity to contagious patients, i.e. all air in patients rooms, intensive care units, etc. during care for COVID-19 patients41. This may be even more important concerning patients in earlier phases of disease, in which contagiousness may be high. This includes both symptomatic and asymptomatic SARS-CoV-2 infected persons in any confined space, such as homes, public transportation, restaurants, etc. The presented findings indicate airborne dissemination of SARS-CoV-2, especially considering the distance SARS-CoV-2 RNA was dispersed. However, further investigations, preferably discriminating between patients in early and later phases of SARS-CoV-2 disease as well as direct sampling of expiratory air from COVID-19 patients will be needed to resolve this question.