Operative Protocol for Testing the Efficacy of Nasal Filters in Preventing Airborne Transmission of SARS-CoV-2.

BACKGROUND: Standardized methods for testing Viral Filtration Efficiency (VFE) of tissues and devices are lacking and few studies are available on aerosolizing, sampling and assessing infectivity of SARS-CoV-2 in controlled laboratory settings. NanoAg-coated endonasal filters appear a promising aid for lowering viable virus inhalation in both adult and younger populations (e.g., adolescents). OBJECTIVE: to provide an adequate method for testing SARS-CoV-2 bioaerosol VFE of bio-gel Ag nanoparticles endonasal filters, by a model system, assessing residual infectivity as cytopathic effect and viral proliferation on in vitro cell cultures. METHODS: A SARS-CoV-2 aerosol transmission chamber fed by a BLAM aerosol generator produces challenges (from very high viral loads (105 PFU/mL) to lower ones) for endonasal filters positioned in a Y shape sampling port connected to a Biosampler. An aerosol generator, chamber and sampler are contained in a class II cabinet in a BSL3 facility. Residual infectivity is assessed from aliquots of liquid collecting bioaerosol, sampled without and with endonasal filters. Cytopathic effect as plaque formation and viral proliferation assessed by qRT-PCR on Vero E6 cells are determined up to 7 days post inoculum. RESULTS: Each experimental setting is replicated three times and basic statistics are calculated. Efficiency of aerosolization is determined as difference between viral load in the nebulizer and in the Biosampler at the first day of experiment. Efficiency of virus filtration is calculated as RNA viral load ratio in collected bioaerosol with and without endonasal filters at the day of the experiment. Presence of infectious virus is assessed by plaque forming unit assay and RNA viral load variations. CONCLUSIONS: A procedure and apparatus for assessing SARS-CoV-2 VFE for endonasal filters is proposed. The apparatus can be implemented for more sophisticated studies on contaminated aerosols.

The role of outdoor and indoor air quality in the spread of SARS-CoV-2: Overview and recommendations by the research group on COVID-19 and particulate matter (RESCOP commission).

There are important questions surrounding the potential contribution of outdoor and indoor air quality in the transmission of SARS-CoV-2 and perpetuation of COVID-19 epidemic waves. Environmental health may be a critical component of COVID-19 prevention. The public health community and health agencies should consider the evolving evidence in their recommendations and statements, and work to issue occupational guidelines. Evidence coming from the current epidemiological and experimental research is expected to add knowledge about virus diffusion, COVID-19 severity in most polluted areas, inter-personal distance requirements and need for wearing face masks in indoor or outdoor environments. The COVID-19 pandemic has highlighted the need for maintaining particulate matter concentrations at low levels for multiple health-related reasons, which may also include the spread of SARS-CoV-2. Indoor environments represent even a more crucial challenge to cope with, as it is easier for the SARS-COV2 to spread, remain vital and infect other subjects in closed spaces in the presence of already infected asymptomatic or mildly symptomatic people. The potential merits of preventive measures, such as CO2 monitoring associated with natural or controlled mechanical ventilation and air purification, for schools, indoor public places (restaurants, offices, hotels, museums, theatres/cinemas etc.) and transportations need to be carefully considered. Hospital settings and nursing/retirement homes as well as emergency rooms, infectious diseases divisions and ambulances represent higher risk indoor environments and may require additional monitoring and specific decontamination strategies based on mechanical ventilation or air purification.

SARS-CoV-2 RNA Recovery from Air Sampled on Quartz Fiber Filters: A Matter of Sample Preservation?

The airborne route of transmission of SARS-CoV-2 was confirmed by the World Health Organization in April 2021. There is an urge to establish standardized protocols for assessing the concentration of SARS-CoV-2 RNA in air samples to support risk assessment, especially in indoor environments. Debates on the airborne transmission route of SARS-CoV-2 have been complicated because, among the studies testing the presence of the virus in the air, the percentage of positive samples has often been very low. In the present study, we report preliminary results on a study for the evaluation of parameters that can influence SARS-CoV-2 RNA recovery from quartz fiber filters spotted either by standard single-stranded SARS-CoV-2 RNA or by inactivated SARS-CoV-2 virions. The analytes were spiked on filters and underwent an active or passive sampling;then, they were preserved at −80 °C for different numbers of days (0 to 54) before extraction and analysis. We found a mean recovery of 2.43%, except for the sample not preserved (0 days) that showed a recovery of 13.51%. We found a relationship between the number of days and the recovery percentage. The results presented show a possible issue that relates to the quartz matrix and SARS-CoV-2 RNA recovery. The results are in accordance with the already published studies that described similar methods for SARS-CoV-2 RNA field sampling and that reported non-detectable concentrations of RNA. These outcomes could be false negatives due to sample preservation conditions. Thus, until further investigation, we suggest, as possible alternatives, to keep the filters: (i) in a sealed container for preservation at 4 °C;and (ii) in a viral transport medium for preservation at a temperature below 0 °C.

Evaluation of Residual Infectivity after SARS-CoV-2 Aerosol Transmission in a Controlled Laboratory Setting.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is mainly transmitted through respiratory droplets, aerosols, or direct contact with fomites from an infected subject. It has been reported that SARS-CoV-2 is stable and viable in aerosol up to 16 h in controlled laboratory conditions. However, the aerosolization conditions varied a lot between the studies. In this work, an experimental laboratory model of SARS-CoV-2 aerosolization was established, employing an impinger nebulizer, a cylindrical chamber for aerosol travel, and a SKC biosampler for the collection of particles. The efficiency of the system was assessed based on the molecular determination of the viral load in the nebulizer after the aerosolization and in the aerosol collected at the end of the travel. Moreover, the residual infectivity was tested in vitro on the Vero E6 cell line, through the observation of the cytopathic effect (CPE), and the quantification of the viral load in the supernatants at 7 days post inoculation (dpi). A high RNA viral load was found in the SKC biosampler after aerosolization, indicating that it was possible to transport a high virus titer through the 30-cm chamber with all the dilutions (initial 105, 104, 103 plaque forming unit-PFU/mL). At the 7 dpi, an increment of the RNA viral load was determined for the dilutions 105 and 104 PFU/mL tested, while only the initial 105 PFU/mL resulted in visible CPE. Our findings allowed us to achieve the resilience of SARS-CoV-2 in aerosol form, at a concentration comparable to those reported for clinical samples. This mode of transmission should be considered for the mitigation and preventive measures to counteract SARS-CoV-2 spreading.

Molecular detection of SARS-CoV-2 from indoor air samples in environmental monitoring needs adequate temporal coverage and infectivity assessment.

The relevance of airborne exposure to SARS-CoV-2 in indoor environments is a matter of research and debate, with special importance for healthcare low-risk settings. Experimental approaches to the bioaerosol sampling are neither standardized nor optimized yet, leading in some cases to limited representativity of the temporal and spatial variability of viral presence in aerosols. Airborne viral viability moreover needs to be assessed. A study has been conducted collecting five 24-h PM10 samples in a COVID-19 geriatric ward in late June 2020, and detecting E and RdRp genes by RT-qPCR with a Ct between 36 and 39. The viral RNA detection at Ct = 36 was related to the maximal numerosity of infected patients hosted in the ward. Lacking a direct infectivity assessment for the collected samples an experimental model has been defined, by seeding twelve nasopharyngeal swab extracts from COVID-19 positive patients on Vero E6 cells; only the four extracts with a viral load above E+10 viral copies (approximately Ct<24) have been able to establish a persistent infection in vitro. Therefore, the cytopathic effect, a key feature of residual infectivity, could be considered unlikely for the environmental PM10 samples showing amplification of viral RNA at Ct = 36 or higher. A standardization of airborne SARS-CoV-2 long-term monitoring and of environmental infectivity assessment is urgently needed.

Potential role of particulate matter in the spreading of COVID-19 in Northern Italy: first observational study based on initial epidemic diffusion.

OBJECTIVES: A number of studies have shown that the airborne transmission route could spread some viruses over a distance of 2 meters from an infected person. An epidemic model based only on respiratory droplets and close contact could not fully explain the regional differences in the spread of COVID-19 in Italy. On March 16th 2020, we presented a position paper proposing a research hypothesis concerning the association between higher mortality rates due to COVID-19 observed in Northern Italy and average concentrations of PM10 exceeding a daily limit of 50 µg/m3. METHODS: To monitor the spreading of COVID-19 in Italy from February 24th to March 13th (the date of the Italian lockdown), official daily data for PM10 levels were collected from all Italian provinces between February 9th and February 29th, taking into account the maximum lag period (14 days) between the infection and diagnosis. In addition to the number of exceedances of the daily limit value of PM10, we also considered population data and daily travelling information for each province. RESULTS: Exceedance of the daily limit value of PM10 appears to be a significant predictor of infection in univariate analyses (p<0.001). Less polluted provinces had a median of 0.03 infections over 1000 residents, while the most polluted provinces showed a median of 0.26 cases. Thirty-nine out of 41 Northern Italian provinces resulted in the category with the highest PM10 levels, while 62 out of 66 Southern provinces presented low PM10 concentrations (p<0.001). In Milan, the average growth rate before the lockdown was significantly higher than in Rome (0.34 vs 0.27 per day, with a doubling time of 2.0 days vs 2.6, respectively), thus suggesting a basic reproductive number R0>6.0, comparable with the highest values estimated for China. CONCLUSION: A significant association has been found between the geographical distribution of daily PM10 exceedances and the initial spreading of COVID-19 in the 110 Italian provinces.

SARS-Cov-2RNA found on particulate matter of Bergamo in Northern Italy: First evidence.

BACKGROUND: The burden of COVID-19 was extremely severe in Northern Italy, an area characterized by high concentrations of particulate matter (PM), which is known to negatively affect human health. Consistently with evidence already available for other viruses, we initially hypothesized the possibility of SARS-CoV-2 presence on PM, and we performed a first experiment specifically aimed at confirming or excluding this research hyphotesys. METHODS: We have collected 34 PM10 samples in Bergamo area (the epicenter of the Italian COVID-19 epidemic) by using two air samplers over a continuous 3-weeks period. Filters were properly stored and underwent RNA extraction and amplification according to WHO protocols in two parallel blind analyses performed by two different authorized laboratories. Up to three highly specific molecular marker genes (E, N, and RdRP) were used to test the presence of SARS-CoV-2 RNA on particulate matter. RESULTS: The first test showed positive results for gene E in 15 out of 16 samples, simultaneously displaying positivity also for RdRP gene in 4 samples. The second blind test got 5 additional positive results for at least one of the three marker genes. Overall, we tested 34 RNA extractions for the E, N and RdRP genes, reporting 20 positive results for at least one of the three marker genes, with positivity separately confirmed for all the three markers. Control tests to exclude false positivities were successfully accomplished. CONCLUSION: This is the first evidence that SARS-CoV-2 RNA can be present on PM, thus suggesting a possible use as indicator of epidemic recurrence.

Searching for SARS-COV-2 on Particulate Matter: A Possible Early Indicator of COVID-19 Epidemic Recurrence.

A number of nations were forced to declare a total shutdown due to COVID-19 infection, as extreme measure to cope with dramatic impact of the pandemic, with remarkable consequences both in terms of negative health outcomes and economic loses. However, in many countries a "Phase-2" is approaching and many activities will re-open soon, although with some differences depending on the severity of the outbreak experienced and SARS-COV-2 estimated diffusion in the general population. At the present, possible relapses of the epidemic cannot be excluded until effective vaccines or immunoprophylaxis with human recombinant antibodies will be properly set up and commercialized. COVD-19-related quarantines have triggered serious social challenges, so that decision makers are concerned about the risk of wasting all the sacrifices imposed to the people in these months of quarantine. The availability of possible early predictive indicators of future epidemic relapses would be very useful for public health purposes, and could potentially prevent the suspension of entire national economic systems. On 16 March, a Position Paper launched by the Italian Society of Environmental Medicine (SIMA) hypothesized for the first time a possible link between the dramatic impact of COVID-19 outbreak in Northern Italy and the high concentrations of particulate matter (PM10 and PM2.5) that characterize this area, along with its well-known specific climatic conditions. Thereafter, a survey carried out in the U.S. by the Harvard School of Public Health suggested a strong association between increases in particulate matter concentration and mortality rates due to COVID-19. The presence of SARS-COV-2 RNA on the particulate matter of Bergamo, which is not far from Milan and represents the epicenter of the Italian epidemic, seems to confirm (at least in case of atmospheric stability and high PM concentrations, as it usually occurs in Northern Italy) that the virus can create clusters with the particles and be carried and detected on PM10. Although no assumptions can be made concerning the link between this first experimental finding and COVID-19 outbreak progression or severity, the presence of SARS-COV-2 RNA on PM10 of outdoor air samples in any city of the world could represent a potential early indicator of COVID-19 diffusion. Searching for the viral genome on particulate matter could therefore be explored among the possible strategies for adopting all the necessary preventive measures before future epidemics start.

Airborne Transmission Route of COVID-19: Why 2 Meters/6 Feet of Inter-Personal Distance Could Not Be Enough.

The COVID-19 pandemic caused the shutdown of entire nations all over the world. In addition to mobility restrictions of people, the World Health Organization and the Governments have prescribed maintaining an inter-personal distance of 1.5 or 2 m (about 6 feet) from each other in order to minimize the risk of contagion through the droplets that we usually disseminate around us from nose and mouth. However, recently published studies support the hypothesis of virus transmission over a distance of 2 m from an infected person. Researchers have proved the higher aerosol and surface stability of SARS-COV-2 as compared with SARS-COV-1 (with the virus remaining viable and infectious in aerosol for hours) and that airborne transmission of SARS-CoV can occur besides close-distance contacts. Indeed, there is reasonable evidence about the possibility of SARS-COV-2 airborne transmission due to its persistence into aerosol droplets in a viable and infectious form. Based on the available knowledge and epidemiological observations, it is plausible that small particles containing the virus may diffuse in indoor environments covering distances up to 10 m from the emission sources, thus representing a kind of aerosol transmission. On-field studies carried out inside Wuhan Hospitals showed the presence of SARS-COV-2 RNA in air samples collected in the hospitals and also in the surroundings, leading to the conclusion that the airborne route has to be considered an important pathway for viral diffusion. Similar findings are reported in analyses concerning air samples collected at the Nebraska University Hospital. On March 16th, we have released a Position Paper emphasizing the airborne route as a possible additional factor for interpreting the anomalous COVID-19 outbreaks in northern Italy, ranked as one of the most polluted areas in Europe and characterized by high particulate matter (PM) concentrations. The available information on the SARS-COV-2 spreading supports the hypothesis of airborne diffusion of infected droplets from person to person at a distance greater than two meters (6 feet). The inter-personal distance of 2 m can be reasonably considered as an effective protection only if everybody wears face masks in daily life activities.