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.

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.