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.

CO2 concentration monitoring inside educational buildings as a strategic tool to reduce the risk of Sars-CoV-2 airborne transmission.

In order to avoid SARS-CoV-2 transmission inside educational buildings and promote the safe reopening of schools, the Italian Government, in line with the other European countries and in accordance with the WHO recommendations, adopted a contingency plan including actions able to guarantee adequate air ventilation in classrooms. Therefore, in this pilot study, a surveillance activity based on the real-time monitoring of CO2 levels as a proxy of SARS-CoV-2 transmission risk, was conducted inside 9 schools (11 classrooms) located in Apulia Region (South of Italy) during the reopening of schools after the lockdown due to COVID-19 pandemic. More specifically, monitoring activities and data treatment were conducted to evaluate the initial scenario inside the classrooms (first stage of evaluation) and the potential improvements obtained by applying a detailed operating protocol of air ventilation based on specific actions and the simultaneous real time visualization of CO2 levels by non-dispersive infrared (NDIR) sensors (second stage of evaluation). Although, during the first evaluation stage, air ventilation through the opening of windows and doors was guaranteed, 6 (54%) classrooms showed mean values of CO2 higher than 1000 ppm and all classrooms exceeded the recommended CO2 concentration limit value of 700 ppm. The development and implementation of tailored ventilation protocol including the real time visualization of CO2 levels allowed to depict better scenariosAn overall improvement of CO2 levels was indeed registered for all classrooms where teachers were compliant and helpful in the management of the air ventilation strategy. Therefore, this study reports the first evidence-based measures demonstrating that, with the exception of few environments affected by structural limits, the real-time visualization and monitoring of CO2 concentrations allowes effective air exchanges to be implemented and contributes to prevent SARS-CoV-2 transmission. Moreover, on the basis of the monitoring outcomes and in order to ensure adequate air ventilation in educational buildings, a 4 level-risk classification including specific corrective actions for each level was provided.

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.

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.