Letter to the Editor. Re: Rufino de Sousa et al., 2022. Detection and isolation of airborne SARS‐CoV‐2 in a hospital setting. Indoor Air, 32 (3), e13023

Dear Editor,

We read the study of Rufino de Sousa et al. ¹ with interest, given the importance of the topic addressed and would like to point out several serious concerns with its content and conclusions.

The findings of the study by Rufino de Sousa et al. ¹ are in contrast with other contemporary evidence, as the authors claim the presence of replication‐competent virus from samples with average Cycle threshold (Ct) values consistently higher than 30 and, in some cases, up to 40 (Figure 1B and caption) using either their hid‐RT‐PCR or their qRT‐PCR methodologies. We recognize the Ct value for the detection of SARS‐CoV‐2 is affected by many factors, including the type of the kit used, the primers used, the targeted genes, the selection of instruments, the operation process, and other factors but the hid‐RT‐PCR method used had excellent agreement with conventional RT‐PCR using the N1 primers based on the adapted method utilized. ²

The authors report that they are “surprised” at finding replication‐competent virus from their air samples, and we would agree with this statement. A comprehensive reporting of the methods is needed to properly evaluate their results from a methodologic perspective, including how potential contamination of viral cultures was excluded. We noted that the specimens were collected from patients in the second week of illness, and if they were immunocompetent hosts, they would not be expected to be shedding infectious virus. ³ , ⁴ , ⁵ Infectious potential declines after Day 8 even among cases with ongoing high viral loads. ⁶ No details are provided about the Ct values of samples taken from patients at the time of collection of the air and environmental specimens, which is essential Supporting Information regarding their capacity to transmit the infection. ⁶

The authors do not report using any media supplemented with antifungal or antibacterial drugs, which is standard practice in most virology laboratory protocols using cell culture. It is well recognized that bacterial and fungal colonies growing on the Vero or other cell lines can produce defects in a monolayer that mimic plaques. Therefore, it seems unusual that 1472 PFUs were reported in Table 3 from the patient room samples, for which 12% (176) were reported to be SARS‐CoV‐2 hid‐RT‐PCR positive under the described methods. The results listed in Table 3 are difficult to interpret and it is possible the methods employed simply picked up SARS‐CoV‐2 RNA dropped onto the plates and were not from replicating virus. No data are provided with respect to the Ct values from these individual samples. In addition, no information is provided as to what the non‐SARS‐CoV‐2 hid‐RT‐PCR‐positive plaques were attributed. The authors did not show enough data, especially direct graphic results, the plaque appearances observed under microscopic examination, and there are multiple missing parameters in their methods. Lednicky et al ⁷ reported virus‐induced cytopathic effects within 2‐day post‐inoculation. However, the amount of virus present in sampled air was low. RT‐PCR for SARS‐CoV‐2 was negative, and three other respiratory viruses were identified: Influenza A H1N1, Influenza A H3N2, and coronavirus OC43. We were also not able to see the use of any positive or negative controls for the air samples. Given the presence of high percentages of SARS‐CoV‐2 hid‐RT‐PCR+ specimens on the floor, bed rails and air vents, it is possible that the THOR device was merely attracting existing RNA in the room and the plaques noted were from fungal or bacterial contaminants and did not truly reflect the presence of replication‐competent SARS‐CoV‐2.

The authors should provide additional levels of support for the presence of infectious virus in these samples, which currently is unconvincing and not supported by the reported Ct values.

Sincerely.

AUTHOR CONTRIBUTION

TJ, DHE, JMC, and CJH contributed to conceptualization, data curation, formal analysis, investigation, methodology, visualization, and writing (review and editing). TJ and DHE contributed to project administration and supervision. TJ contributed to writing (original draft).

CONFLICT OF INTEREST

TJ's competing interests are accessible at: https://restoringtrials.org/competing‐interests‐tomJefferson. CJH holds grant funding from the NIHR, the NIHR School of Primary Care Research, the NIHR BRC Oxford and the World Health Organization for a series of Living rapid review on the modes of transmission of SARS‐CoV‐2 reference WHO registration No2020/1077093. He has received financial remuneration from an asbestos case and given legal advice on mesh and hormone pregnancy tests cases. He has received expenses and fees for his media work including occasional payments from BBC Radio 4 Inside Health and The Spectator. He receives expenses for teaching EBM and is also paid for his GP work in NHS out of hours (contract Oxford Health NHS Foundation Trust). He has also received income from the publication of a series of toolkit books and for appraising treatment recommendations in non‐NHS settings. He is Director of CEBM and is an NIHR Senior Investigator. DE holds grant funding from the Canadian Institutes for Health Research and Li Ka Shing Institute of Virology relating to the development of COVID‐19 vaccines as well as the Canadian Natural Science and Engineering Research Council concerning COVID‐19 aerosol transmission. He is a recipient of World Health Organization and Province of Alberta funding which supports the provision of BSL3‐based SARS‐CoV‐2 culture services to regional investigators. He also holds public and private sector contract funding relating to the development of poxvirus‐based COVID‐19 vaccines, SARS‐CoV‐2‐inactivation technologies, and serum neutralization testing. JMC holds grants from the Canadian Institutes for Health Research on 399 acute and primary care preparedness for COVID‐19 in Alberta, Canada and was the primary local Investigator for a Staphylococcus aureus vaccine study funded by Pfizer for which all funding was provided only to the University of Calgary. He is co‐investigator on a WHO‐funded study using integrated human factors and ethnography approaches to identify and scale innovative IPC guidance implementation supports in primary care with a focus on low‐resource settings and using drone aerial systems to deliver medical supplies and PPE to remote First Nations communities during the COVID‐19 pandemic. He also received support from the Centers for Disease Control and Prevention (CDC) to attend an Infection Control Think Tank Meeting. He is a member and Chair of the WHO Infection Prevention and Control Research and Development Expert Group for COVID‐19 and a member of the WHO Health Emergencies Programme (WHE) Ad hoc COVID‐19 IPC Guidance Development Group, both of which provide multidisciplinary advice to the WHO and for which no funding is received and from which no funding recommendations are made for any WHO contracts or grants. He is also a member of the Cochrane Acute Respiratory Infections Working Group.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in Indoor Air at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9111425/pdf/INA‐32‐0.pdf.