Since the beginning of coronavirus disease 2019 (COVID-19) pandemic, large attention has been focused on the relationship between SARS-CoV-2 diffusion and environment. As a matter of fact, clear evidence of the transmission of SARS-CoV-2 via respiratory aerosol would be of primary importance; at the same time, checking the presence of SARS-CoV-2 in wastewater can be extremely useful to control the diffusion of the disease. Up to now, many studies report SARS-CoV-2 concentrations in indoor/outdoor air samples or water/wastewater samples that can differ by order of magnitude. Unfortunately, complete information about the scientific approach of many studies is still missing, relating to: samplers and sampling materials performances, recovery tests, measurement uncertainty, robustness, detection and quantification limits, infectivity of captured virus, virus degradation during sampling, influence of sample pre-treatments (included freezing) on results, effects of inhibitors, sample alterations due to manipulation, validation of methods and processes, quality assurance according to ISO/IEC 17025 requirements. Based on the first experiences focused on the presence of SARS-CoV-2 in environmental samples such as air quality filters or impingers collection solutions, the present study describes a coherent preliminary approach to SARS-CoV-2 indoor and outdoor air sampling in order to overcome the evident lack of standardization. Three aspects are highlighted here: the first solution to assure quality and consistency to air sampling relies on the development of recovery tests using standard materials and investigating sampling materials, sampling techniques, sampling durations, sample conservation and pre-treatments; secondly, in order to overcome the shortcomings of every single sampling technique, coupling different samplers in parallel sampling could be an efficient strategy to collect more information and make data more reliable; finally, with regards to airborne virus sampling, the results could be confirmed by simplified emission and dilution models.

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The airborne transmission path for SARS-CoV-2 is of primary scientific and health-related interest as it could actually involve management, accessibility, use and functionality of many activities, including hospitals), schools, workplaces, factories, transport, sport venues and outdoor environment. It is necessary to develop a sampling and analytical method for virus-laden bioaerosol that could be considered reliable and validated according to ISO/IEC 17025 requirements. The present paper defines sample pretreatments aiming at recover SARS-CoV-2 from glass-fiber and PTFE filters employed by low and high-volume air samplers. Recovery test results focused on the sample concentration step carried out by means of ultracentrifugation are reported as well. Human coronavirus strain OC43 (a surrogate β-coronavirus with the same SARS-CoV-2 particle structure) was used to validate each step of the recovery tests. We found that the elution efficiency of coronavirus OC43 from glass-fiber and quartz filters could be strongly enhanced by using an elution buffer containing up to 40% of fetal calf serum. Moreover, the recovery from PTFE filters is much higher and easier than from glass-fiber filters: for glass-fiber filters a 3 h-shaking phase, followed by a 30 s-vortexing step, are necessary to elute viral infective particles; for PTFE, 60 min-shaking is enough. The effect of suction time on filters could be resumed as follows: sampling durations up to 20 min at a flow rate of 500 L/min do not affect recovery efficiencies from 10 cm glass-fiber filters, whereas the recovery efficiency of infectious virions from 4.7 cm PTFE filters decreases of a factor 2 after 3 h of sampling at a flow rate of 20 L/min. The recovery efficiency of ultracentrifugation turns out to be around 57%. The effect of storage temperature of filters immersed in a transport medium on coronavirus infectivity is assessed as well. Based on the sampling techniques and the analytical methods developed as described in the present study, many field tests were carried out reporting virus concentrations up to 50 genomic copies per cubic meter of air in domestic environment with poor ventilation condition, whereas in hospital wards the detectable concentrations of SARS-CoV-2 were generally lower than 10 genomic copies per cubic meter of air.

The most recent scientific studies have finally identified the airborne transmission of SARS-CoV-2 as significant. Therefore, the airborne transmission path for SARS-CoV-2 is of primary scientific and health-related interest as it could actually involve management, accessibility, use and functionality of many activities, including hospitals (where COVID wards represent only a part of the critical issues), schools, workplaces, offices, factories, means of transport, sports venues, and the outdoor environment. It is necessary to develop a sampling and analytical method for virus-laden bioaerosol that could be considered reliable and validated according to ISO/IEC 17025 requirements.The present paper defines samples pretreatments aiming at recover SARS-CoV-2 from glass-fiber and PTFE filters used by low and high-volume air samplers. Recovery test results focused on the sample concentration step carried out by means of ultracentrifugation are reported as well. Human coronavirus strain OC43 (a surrogate β-coronavirus with same SARS-CoV-2 particle structure) was used to validate each step of the recovery tests.We obtained the following results:-the recovery efficiency from glass-fiber filters and quartz filters could be strongly enhanced by using an elution buffer containing up to 40% of fetal calf serum.-the recovery efficiency of coronavirus OC43 (HCoV-OC43) from PTFE filters is much higher and easier than from glass-fiber filters; for glass-fiber filters, we found that a two-step procedure is necessary to elute viral infective particles: a 3 hour-shaking step, followed by a 30 seconds-vortexing step. For PTFE 60 minutes-shaking is enough.-the effect of suction time on filters could be resumed as follows: concerning 10cm glass-fiber filters, sampling durations up to 20 minutes at a flow rate of 500 L per minute do not affect recovery efficiencies. On the contrary, the recovery efficiency of infectious virions from 4.7cm PTFE filters decreases of a factor 2 after 3 hours of sampling at a flow rate of 20 L per minute.-the recovery efficiency of ultracentrifugation is 57%.Furthermore, the effect of the storage temperature of the filters immersed in the transport medium on the infectivity of HCoV-OC43 has been assessed.Based on the sampling techniques and the analytical methods developed as described in the present study, many field tests were carried out reporting virus concentrations up to 50 genomic copies per cubic meter of air in domestic environment with poor ventilation condition, whereas in hospital wards the detectable concentrations of SARS-CoV-2 were generally lower than 10 genomic copies per cubic meter of air.The developed methods, aiming at providing the community with reliable determinations about the presence of SARS-CoV-2 and other airborne pathogens in air, prove essential for the development, during the pandemic, of a coherent management of places (especially of crowded ones) such as means of transport, stations, gyms, theaters, cinemas.

Since the beginning of coronavirus disease 2019 (COVID-19) pandemic, large attention has been focused on the relationship between SARS-CoV-2 diffusion and environment. As a matter of fact, clear evidence of the transmission of SARS-CoV-2 via respiratory aerosol would be of primary importance; at the same time, checking the presence of SARS-CoV-2 in wastewater can be extremely useful to control the diffusion of the disease. Up to now, many studies report SARS-CoV-2 concentrations in indoor/outdoor air samples or water/wastewater samples that can differ by order of magnitude. Unfortunately, complete information about the scientific approach of many studies is still missing, relating to: samplers and sampling materials performances, recovery tests, measurement uncertainty, robustness, detection and quantification limits, infectivity of captured virus, virus degradation during sampling, influence of sample pre-treatments (included freezing) on results, effects of inhibitors, sample alterations due to manipulation, validation of methods and processes, quality assurance according to ISO/IEC 17025 requirements.Based on the first experiences focused on the presence of SARS-CoV-2 in environmental samples such as air quality filters, air-liquid impingers and wastewater samples, the present study describes a coherent preliminary approach to SARS-CoV-2 environmental sampling in order to overcome the evident lack of standardization. Three aspects are highlighted here: the first solution to assure quality and consistency to environmental sampling relies on the development of recovery tests using standard materials and investigating sampling materials, sampling techniques, sampling durations, sample conservation and pre-treatments; secondly, in order to overcome the shortcomings of every single sampling technique, coupling different samplers in parallel sampling could be an efficient strategy to collect more information and make data more reliable, in particular for air samples; finally, with regards to airborne virus sampling, the results could be confirmed by simplified emission and dilution models.