key: cord-0706141-b33uva1m authors: Pradillo, Beatriz Linillos; Rancan, Lisa; Ramiro, Elías Díaz; Vara, Elena; Artíñano, Begoña; Arias, Javier title: Determination of SARS-CoV-2 RNA in different particulate matter size fractions of outdoor air samples in Madrid during the lockdown date: 2021-02-17 journal: Environ Res DOI: 10.1016/j.envres.2021.110863 sha: 7050050b8573069d27131987ad779075d8a25065 doc_id: 706141 cord_uid: b33uva1m BACKGROUND: Previous studies described the presence of SARS-CoV-2 in outdoor air particulate matter (PM) in urban areas of northern Italy and USA. The city of Madrid was heavily affected by COVID-19 during March-June 2020. Also, this city usually displays high concentrations of PM under several atmospheric situations. This is mandatory to assess the presence of viral RNA in PM, as an indicator of epidemic recurrence. Our study was aimed at investigating the presence of SARS-CoV-2 RNA in outdoor air samples (on PM10, PM2.5 and PM1). METHODS: six samples of PM10, PM2.5 and PM1 were collected between the May 4(th) and 22(nd) 2020 in Madrid, on quartz fiber filters by using MCV high volume samplers (30 m(3) h(−1) flow) with three inlets (Digitel DHA-80) for sampling PM10, PM2.5 and PM1. RNA extraction and amplification was performed according to the protocol recently set by Setti et al., 2020c in Italy. Up to three highly specific molecular marker genes (N1, N2, and RP) were used to test the presence of SARS-CoV-2 RNA. RESULTS: After RNA extraction and expression measurements of N1, N2 and RP genes from all the collected filters, no presence of SARS-CoV-2 RNA was observed. Control test to exclude false positive results were successfully accomplished. CONCLUSIONS: No presence of SARS-CoV-2 in quartz fiber filters samplers for PM10, PM2.5 and PM1 fractions was observed in our study carried out in Madrid during the month of May 2020. Nevertheless, the absence of viral genomes could be due to different factors including: limited social interactions and economic activities resulting in reduced circulation of the coronavirus, lower daily PM concentration in outdoor air, as well as to meteorological stability and higher temperature that characterize spring season. Further researches should be carried out during the winter, in presence of higher viral circulation and daily PM exceedances. On 31 December 2019, the World Health Organization was informed of a disease caused by the SARS-CoV-2 virus in China, which has spread from Country to Country, becoming a global pandemic (Lu et al., 2020) along the year 2020 As a viral respiratory infectious disease, the possible ways of transmission between humans are described through direct (person-to-person) and indirect (via fomites) contact (Asadi et al., 2020; Morawska and Cao, 2020) . Setti et al. (Setti et al., 2020d) , hypothesised that the SARS-CoV-2 virus could be present in particulate matter (PM), with the airway being the third transmission mechanism (World Health Organization, 2020 ). It seems that high levels of urban air pollution, weather and specific climate conditions have a significant impact on the increased rates of confirmed COVID-19 total number, daily new and total deaths cases, possible attributed not only to indoor but also to outdoor airborne bioaerosols distribution (Zoran et al., 2020) . In the case of other viruses (Ma et al., 2017; Sedlmaier et al., 2009; Srensen et al., 2000; Zhao et al., 2019) , it has been shown that particulate matter can act as a 'carrier' for viral droplet nuclei, causing further spread of viral infection. Qing et al. (Qing et al., 2016) showed that RSV (respiratory syncytial virus) infection was favoured by particle transport after a positive correlation between infection rate and PM2.5 (particles with aerodynamic diameter < 2.5 µm) and PM10 (particles with aerodynamic diameter < 10 µm) particle fractions. On the other hand, Cheng et al. (Cheng et al., 2020) showed that an increase in PM2.5 equal to 10 µg/m 3 was significantly associated with a higher incidence of measles in 21 Chinese cities. Also, Peng et al. (Peng et al., 2020) showed that high levels of PM concentrations significantly affected the spread of measles in Lanzhou, China. Previous studies considered an important role of airborne transmission causing the abnormal outbreaks of COVID-19 observed in northern Italy and the United States (Setti et al., 2020a) . The Harvard Wu School of Public Health and others (Wu et al., 2020) , has suggested a strong association in the United States between the increase in PM concentration and mortality rates from J o u r n a l P r e -p r o o f COVID-19. The Italian Society of Environmental Medicine (SIMA) described a possible relationship between the high mortality rates observed in northern Italy due to COVID-19 and PM concentrations (Setti et al., 2020b) . A significant correlation was also found between the geographical distribution of daily PM10 excesses in 110 Italian provinces (number of days exceeding the PM10 law limit of 50 µg/m 3 ) and the spread of COVID-19 infection before the lockdown imposed by the Italian Government. The presence of SARS-CoV-2 RNA in 34 PM10 samples in the outdoor air of an industrial site in the province of Bergamo, which in March 2020 was the epicentre of the Italian epidemic, seems to confirm (at least in the case of atmospheric stability and high PM concentrations) that the virus can create clusters with the particles and be transported and detected in PM10 (Setti et al., 2020c and 2020d) . Spain has been a very affected country since the beginning of the pandemic. In the study period, between 4 th and 22 nd of May 2020, were reported 250,287 confirmed cases by COVID-19. Its capital, Madrid, accounted for 28.5% of the total cases in Spain (Informe nº 33. Análisis de los casos de COVID-19 notificados a la RENAVE hasta el 10 de mayo en España a 29 de mayo de 2020. Equipo COVID-19. RENAVE. CNE. CNM (ISCIII)). However, a daily decrease in the number of confirmed cases and the number of deaths was observed in Madrid in that same period ( Sampling dates and periods are shown in Table 1 . Sampling protocol was made according that standard for PM measurement in ambient air with special ad-hoc features. Before starting with the sampling process, Whatman Quartz fiber filters (150 mm diameter and QMA quality) were baked at 450 °C for 24 hours. Filters were then placed in the inlet directly from the oven. Finally, six samples of each PM10, PM2.5 and PM1, were collected between 4 th and 22 nd of May 2020. After sampling, filters were, directly from the inlet, ultrafrozen with liquid nitrogen and dry ice in Falcon tubes to be transported to the laboratory for analysis (Department of Biochemistry and Molecular Biology, UCM). Field blank filters were collected and analyzed with each batch of samples in order to establish the artifacts due to adsorption of constituents into quartz filter during and after sampling. The described procedure was used for J o u r n a l P r e -p r o o f the blank filters, which were placed in the inlets, but no sampling was performed in these cases. Inlets were dismounted and cleaned with 70% ethanol before each sampling, taking care of specific cleaning of the inlet holes and pins. Personnel involved in the sampling were wearing personal protective equipment (PPE): robes, gloves and masks, during the sampling. In the laboratory, RNA was extracted following the protocol described by Setti et al. (Setti et al., 2020c) . Given the "environmental" nature of the sample, presumably rich in DNA and RNA polymerase inhibitors, we proceeded to extract RNA using the rapid RNA fecal soil microbe kit adapted to the type of filters (Zymoresearch Ldt, 2020 Cat #R2040). Half filter was rolled, with the top side facing inward, in a 5 ml polypropylene tube, together with the beads provided in the kit. From the initial 1 ml of lysis buffer, we were able to get about 400 μl of solution, which was then processed as defined by the standard protocols, resulting in a final eluate of 15 μl. Subsequently, 6 μl were used for the SARS-CoV-2 testing using Efficient 2019-nCOV detection kit, one step RT-qPCR (AnyGenes, Paris Cat·#19nCoVd-100) and following the manufacturer's instructions for our amplification system (7500 Fast, Applied Biosystems). The probes and primers included in the detection kit specifically recognize two regions of the COVID-19 nucleocapsid (N1 and N2 genes) and also control human RNase P (RP gene) (species control, recommended by CDC, Centers for disease control and prevention). It also includes a positive control CTR-POS for quality control and a second positive control (CTR-HSC), as a control of RNA purification. Therefore, up to three highly specific molecular marker genes (N1, N2, and RP) were used to test the presence of SARS-CoV-2 RNA on particulate matter. Meteorological conditions in Madrid during the 2-months-period (April-May 2020) were analyzed from the station located at the sampling site in the CIEMAT facilities. Table 1 shows mean values of these concentrations and meteorological parameters during the samplings. The month of April, was in general rainy and windy, typical of the Madrid spring weather, normally affected by the frequent pass of frontal systems. There were some precipitation events and relative humidity was high during this This is a common and well documented phenomenon that, under specific synoptic patterns, contributes to increase the ambient levels of particulate matter in Spain (Salvador et al., 2014 (Salvador et al., , 2013 . These two events took place the 4 th of May and during the period between the 7 th and the 9 th of May. Consequently, the coarse fraction of particulate matter (PM10) concentrations experienced a visible increase during these periods (Figure 2 Regarding the molecular analysis of the PM10, PM2.5 and PM1 filters, we first carried out the control for the quality controls. The expected results for the quality controls are shown in the following In our study we observed the following results for quality controls, shown in Confronting the results of the controls with those expected, we could affirm that the experiment gone well and that the technique was carried out correctly. Regarding the results of the samples, interpretation following the manufacturer's instructions is shown in Table 4 . It should be noted that (Setti et al., 2020c) . They collected the PM10 filters before the Italian government declared total confinement to its population. The filters were then stored for at least four weeks before the molecular genetic analysis (Setti et al., 2020c) . A similar situation occurred in Madrid, where high concentrations of PM particles exceeded for several days the PM10 daily limit value of 50 μg/m 3 before the pandemic lockdown (even if, in contrast with the north of Italy, the limit values were never exceeded during 2018) and where a high COVID-19 presence was observed too. However, in Madrid, during the pandemic lockdown, the limit values of pollution were never exceeded. On March 14 th , Spain declared a state of alarm and the lockdown situation until April 28 th . In these conditions, only essential and primordial activities could be carried out. From April 28 th until the end of May, Madrid was in phase 0 (beginning phase towards the new normality and relaxation of the isolation measures). During this period, the activity in some works was restored, the stored began to open by previous appointment and people were allowed to go out with time limitation and following a time schedule. These activities began to increase the levels of atmospheric pollutants, being noticed a difference between the first days of May and the end of the month (Fig. 2a and 2b) , also enhanced by the precipitation events and general unstable meteorological conditions. Once the atmospheric conditions became more stable, the study began. Hence, the filters were obtained from May 4 th to May 21 st . The filters were J o u r n a l P r e -p r o o f collected at the beginning of phase 0 and were analyzed simultaneously to their collection. It should be mentioned that in the filters collected from the 4 th and the period between the 7 th and 9 th of May, there was presence of Saharan dust. This is an important event since dust storms are very effective for the propagation of several kind of viruses. In the case of the influenza A virus, it was shown that when the levels of Asian dust particles were significantly higher than the average ones, there was greater spread (Chen et al., 2016) . However, in our study, during this period, the three investigated genes were negative suggesting that there was no presence of SARS-CoV-2 RNA. It should be taken into account that some factors could also have affected the identification RNA from the virus in the PM10, PM2.5 and PM1 filters collected in May (Table 5) in Madrid. First, the limitations of socioeconomic activity which led to the low atmospheric pollution contamination levels (lower daily particulate matter means values in outdoor air). This is also reinforced by the sampling site, which is located at an urban background area near the university with no academic activity during the sampling period and therefore low pedestrian flow. This was reflected in a fifty percent reduction in the cumulative incidence rate since the beginning of the study compared to the end of May in the Moncloa-Aravaca district ( Fig. 3a and 3b) . Finally, the meteorological conditions that cleaned up the atmosphere in the period before the sampling could have helped reducing the PM as well as the virus concentrations. In addition to highlighting the meteorological stability and higher temperature that characterize spring season. Our results support the idea that, to be able to identify the SARS-CoV-2 RNA in outdoor samples both high PM levels and high have to occur at the same time. This is in accordance with previous studies that and health outcomes observed, as occurred in northern Italy and the USA (Setti et al., 2020c; Tung et al., 2021; Zoran et al., 2020) . On this regard, the search for viral genomes in particles could be considered as a preventive strategy for future epidemics and strategies for the reduction of PM emitted by some anthropogenic sources. This would allow to mitigate the exposure of citizens to PM and uncontrolled aerosols, which is well known that cause many negative health effects. In relation with COVID-19, air pollution could influence its progression by increasing host susceptibility to viral infections and by independently increasing the risk of cardiovascular complications, chronic obstructive pulmonary disease (EPOC), and other conditions that increase the severity of viral infections (Setti et al., 2020b; Zoran et al., 2020) . However, more research needs to be done to know the role of ambient air pollution in the spread of this virus. Particularly in Madrid, it would be interesting in the near future to measure again the presence of SARS-CoV-2 RNA in the PM filters. This is due to the increase in the number of COVID-19 cases observed in the city after the partial recovery of the economic-social activity that also led to an increase of contamination under different atmospheric and meteorological situations (Tellier et al., 2019 ). An additional step would be to investigate not only the presence but the possible virulence of SARS-CoV-2 present in the particles to see if the virus can remain vital and infectious for a defined time in the outside particles (Ficetola et al., 2020.; van Doremalen et al., 2020) . No presence of SARS-CoV-2 in quartz fiber filters samplers for PM 10, Peng, L., Zhao, X., Tao, Y., Mi, S., Huang, J., Zhang, Q., Peng, L., 2020. The effects of air pollution and meteorological factors on measles cases in Lanzhou , China 13524-13533. Qing, Y., JunFen, F., JianHua, M., ShiQiang, S., 2016. Haze is a risk factor contributing to the rapid spread of respiratory syncytial virus in children. J o u r n a l P r e -p r o o f The coronavirus pandemic and aerosols: Does COVID-19 transmit via expiratory particles? 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