key: cord-0967727-w5px1j61
authors: Pivato, A.; Amoruso, I.; Formenton, G.; Di Maria, F.; Bonato, T.; Vanin, S.; Marion, A.; Baldovin, T.
title: Evaluating the presence of SARS-CoV-2 RNA in the particulate matters during the peak of COVID-19 in Padua, northern Italy
date: 2021-04-16
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.147129
sha: a97ce427156c222df866b1d076cacf0a29278c4e
doc_id: 967727
cord_uid: w5px1j61

The airborne transmission of SARS-CoV-2, the etiologic agent of the current COVID-19 pandemic, has been hypothesized as one of the primary routes of transmission. Current data suggest a low probability of airborne transmission of the virus in open environments and a higher probability in closed ones, particularly in hospitals or quarantine facilities. However, the potential diffusion of the virus in open environments, especially using particulate matter (PM) as a transport carrier, generated concern in the exposed populations. Several authors found a correlation between the exceeding of the PM10 concentration limits in some Italian cities and the prevalence of Covid-19 cases detected in those areas. This study investigated the potential presence of SARS-COV-2 RNA on a representative series of PM samples collected in the province of Padua in Northeastern Italy during the first wave of COVID pandemic. Forty-four samples of PM2.5 and PM10 were collected between February 24 and March 3, 2020 and analyzed with RT-qPCR for SARS-CoV-2 RNA. The experimental results did not indicate the presence of SARS-CoV-2 RNA in the outdoor PMs, thus confirming the low probability of virus airborne transmission through PM.

Airborne transmission has been recognized as one of the primary routes of conveyance of etiologic agents such as respiratory viruses, including the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome coronaviruses (Booth et al., 2005; Yu et al., 2004; Tellier et al., 2019) . SARS-CoV-2, the cause of the current COVID-19 pandemic, also falls into this category (Lewis, 2020; National Research Council, 2020; WHO, 2020; Prather et al., 2020) . Recently, 239 scientists from 32 countries have written an open letter to the World Health Organization (WHO) emphasizing the importance of preventing its airborne transmission (Morawska and Milton, 2020) .

Most credited SARS-CoV-2 transmission pathway is by respiratory droplets as small as 5 μm or larger, generated by sneezes, coughs, or breaths during normal speaking (Lewis, 2020; National Research Council, 2020; Yu et al., 2018; WHO, 2020c) . The airborne lifetime of the droplets and the range of transmission (e.g. more than 1 m) remains unclear (Anderson et al., 2020; Morawska and Cao, 2020) . The mechanisms underlying the airborne transport of SARS-CoV-2 have not been fully elucidated. Also, the influence of the carrier typology (e.g., droplets and aerosols including particulate matter, PM), the role of environmental conditions (e.g., wind speed, temperature, humidity, UV radiations, seasonal allergens such as pollens and spores), and air pollutant concentrations, remain unclear.

A recent study suggests a low probability of airborne virus transmission in open environments and a higher one in closed ones, especially in hospitals or quarantine facilities (Contini and Costabile, 2020) . However, the experimental evidence supporting the statement above is weak. It mainly focuses on aerosols and droplets produced by infected patients through coughing, sneezing, speaking, and breathing. The presence of SARS-CoV-2 in the aerosols sampled inside two Hospitals of Wuhan during pandemic peaks was observed by Liu et al. (2020) . Santarpia et al. (2020) The concern about the diffusion of the virus in open environments, particularly using PMs as carriers, is still widespread in the population. Some studies (Cascetta et al., 2021; Coccia 2020; Bontempi 2020; Setti et al. 2020a ) found a correlation between the exceeding of the PM10 concentration limits in some Italian cities and the number of Covid-19 cases. Despite this limited evidence and bearing in mind that correlation is not causation (Altman and Krzywinski, 2015) , the cause-and-effect relationship between PM concentration and COVID-19 prevalence and symptom severity remains controversial (Anand et al., 2021) In this context, PMs may act as physical carriers of the virus, as possible infection boosting factors (Comunian et al., 2020; Paital and Agrawal, 2020) , or as a combination of both. These possibilities require further investigation and proper experimental studies.

Preliminary research on the relationship between PMs and virus transmission was carried out by Setti et al. (2020b) . It reported a first preliminary detection of the presence of SARS-COV-2 RNA on the PM from examining 34 PM10 samples collected from an industrial site in the province of Bergamo in Northern Italy.

On the other hand, other outdoor air samples were simultaneously collected in Venice in Northeastern Italy and Lecce in Southern Italy in May 2020 and they were tested negative for SARS-CoV-2 RNA (Chirizzi et al., 2021) . In these works, the hypothesized mechanism is that virus-laden aerosol could interact with the preexisting atmospheric particles creating clusters of carriers (Belosi et al., 2021) .

Due to the contradictory results previously mentioned and the lack of studies on this topic, this project aims to further investigate the potential presence of SARS-CoV-2 RNA on a representative series of PM collected in the Province of Padua in Northeastern Italy, an area severely affected by the first wave of the COVID-19 pandemic. The methodological issues related to the extraction and detection of viral RNA are also analyzed and discussed.

J o u r n a l P r e -p r o o f Journal Pre-proof

Since the initial spreading of the pandemic wave (February-March 2020), Italy has been recognized as one of the most affected countries. In response to the uncontrolled increase of COVID-19 cases (Figure 1 ), the Italian government imposed several restrictions (lockdown, compulsory usage of sanitary masks, etc.).

Finally, on May 17, 2020, the nationwide lockdown ended, and less strict measures were adopted locally.

PM sampling was performed in the Province of Padua (Figure 2 ) with the frequency reported in Table 1 between February 24 and March 9, 2020, i.e. the two weeks before lockdown. The sampling sites are described in Table 2 and classified according to the European Directive 2008/50/EC. During the sampling period (14 days), the meteorological conditions were registered from dedicated stations installed directly in the PM samplers or from the closest stations (Table 1) . Considering the collected samples, the average daily temperature was 7.9 •C (Standard Deviation, SD=1.0); the average daily irradiation was 99.7 W/m 2 (SD=62.6); the average daily wind density was 1.2 m/s (SD=0.5). Precipitations were observed only for 14 samples.

PM (PM10, PM2.5) samples were collected on quartz fiber filters (47 mm Ø, Whatman QMA, GE Healthcare, USA) using the low-volume sampling setting according to the European standard EN 12341:2014 at a nominal flow of 2.3 m 3 h −1 for 24 h, starting at midnight. The filters have a retention efficiency higher than 99.95% for particles with an aerodynamic diameter of 0.3 µm. Before reaching the laboratory, the samples remained at the sampling station from 3 to 4 days in containers kept in the dark and at 20 °C. Then, the filters were conditioned for gravimetric analysis for 48 h in a chamber with constant temperature 20 ± 1°C and relative humidity 50 ± 5% (Emerson S05KA Emerson Network Power, Italy). The filters were then weighed twice with an analytical balance with a sensitivity of 0.0001 mg (Sartorius series Genius, mod. SE2, Germany). The final weight was calculated as the average of the two measurements.

Finally, the samples were frozen in clean Petri slides at −20°C for the subsequent analysis. Laboratory testing was performed by the BSL-2 Research Laboratory of Hygiene and Applied Microbiology of the Padua J o u r n a l P r e -p r o o f Journal Pre-proof University (Italy). The laboratory implements updated OECD Good Laboratory Practices and adopted fundamental precautions for the correct handling of RNA samples.

Recovery of PM from quartz fiber filters reprised the procedure described by Roper et al. (2015) . Filters were placed with the PM face down in 100 mL glass beakers containing 5 mL of a 9:1 methanol/sterile distilled water solvent. Beakers were then sonicated for 2 min in a water bath sonicator at 50 KHz (Labsonic LBS1, Falc Instruments, Italy). Reported PM removal efficiency following sonication is of 98.0±1.4%. The solvent was then collected in a 15 mL Falcon® conical centrifuge tube and the filter was sonicated again, repeating the described step. After the second round of sonication, both filter and beaker were rinsed with 5 mL of clean solvent, that ultimately was also collected in the same 15 mL tube. Falcon tubes were then centrifuged at 5500 g for 15 min (refrigerated centrifuge Allegra 21R, Beckman Coulter, California, USA) to separate solids from the liquid solvent. Subsequently, the two phases were processed in parallel to detect viral particles both complexed to the pellet PM or still suspended in the supernatant. Retention efficiency for particles with molecular weight similar to the SARS-CoV-2 virion is of >90%, as per manufacturer's specifications.

RNA extraction from concentrated supernatant was carried out on a volume of 140 µL with a commercial kit (QIAamp viral RNA mini kit, Qiagen, Germany), following the manufacturer's instructions. RNA extraction from the pellet was performed with the same total RNA extraction kit (QuickRNA TM Fecal/Soil Microprep Kit R2040, Zymo Research, USA) used by Setti et al. (2020 b) . RNA extraction efficiency for both kits, in terms of J o u r n a l P r e -p r o o f Journal Pre-proof RNA yield, is reported to be >90% by the respective manufacturers. The pellet was resuspended in 600 µL of the kit RNA lysis buffer. It was then transferred into the provided 2 mL bashing beads tube and thoroughly vortexed for 60 sec. Apart from these minor modifications, extraction proceeded according to the manufacturer's protocol. An Internal Positive Control (IPC), i.e. 3 µL (9 x 10 4 gc/µL) of synthetic SARS-CoV-2 armored (i.e. encapsidated) RNA (2019-nCoV E gene aRNA kit cod. 001B-03886, EVAg-Protisvalor, France), was added to each sample before extraction as process indicator. IPC was also used to assess the presence of inhibitors of the quantitative reverse-transcription polymerase chain reaction (RT-qPCR).

Two WHO-shared One-step RT-qPCR assays were chosen for the molecular detection of SARS-Cov-2 RNA, targeting genes N (screening) and ORF1b-nsp14 (confirm) . Primers and dual-labeled probes were provided from Thermo Fisher (USA). Synthetic dsDNA fragments were used as positive controls and were also purchased by GeneArt/Thermo Fisher. In each PCR run, 2 replicates were loaded for each extract. Moreover, 2 positive and 2 negative controls were included. Amplification of the IPC was carried out with the dedicated assay (i.e. primers and dual-labeled probe), also provided with the aRNA kit, following the manufacturer's instructions. PCR runs were carried out on a StepOne-Plus™ Real-Time PCR System (Applied Biosystems, USA). Positivity was attributed only to reactions with cycle threshold (Ct) <40. The limit of detection (LOD) of the implemented assays was determined using DNA plasmids as positive standards and found to be below 10 genome copies (gc) per reaction (i.e. sample volume / well = 4 µL), that is. 2.5 gc/µL. Nevertheless, some authors suggested a possible differential performance between the N and the Orf1b assay, with N showing a 10x sensitivity in both clinical and environmental samples Baldovin et al., 2020) . The above described processes are graphically represented in the flow chart of Figure 3 .

Regarding the second aspect, the considerable atmospheric residence time (days to weeks) of PM before sampling dominates the nucleic acid persistence because, in this period, the cluster of particulate and virus could be primarily influenced by meteorological parameters, such as UV radiation, temperature, and oxidizing agents like NO X and ozone. This scenario is particularly relevant in the Province of Padua, which is characterized by low wind speed accompanied by long periods of stable conditions with shallow mixing layers, especially during the winter period. Therefore, it is also unlikely that the virus will stay viable in these conditions. Moreover, considering other parameters, such as SARS-CoV-2's viability, infectivity, and infective dose, which remain unclear (Barakat et al. 2020) , it can be concluded that the outdoor airborne transmission is much less probable than the indoor route.

In conclusion, based on the experimental results and the above-reported observations, we believe that monitoring for the presence of SARS-CoV-2 RNA in outdoor particulates is not suitable for an efficient early indicator of SARS-Cov-2 diffusion or/and an early indicator of a recurrence of the pandemic. Table 1 . Information of the PM samples: date of sampling; sample code (sample code used in the regional monitoring network); sampling site, including the referred codes used in Figure 2 ; meteorological conditions; PM typology (PM2.5 or PM10); PM concentration. J o u r n a l P r e -p r o o f Table 3 . Comparison from the current study and the ones of Chirizzi et al. (2021) and Setti et al. (2020 b) Operative Figure 1 . The development of COVID-19 in Italy and the Veneto Region when the PM samples were collected. The graph was based on Gatto et al. (2020) . Time marks (A, B, C, and D) represent the most critical epidemiological events and measures for both mobility and contact restrictions at each time point: A) On February 21, 2020 (day 1), "patient one" was officially confirmed as a case of COVID-19 by the "Ospedale Sacco" in Milan; by the end of the day, other 14 cases in Lombardy and 2 cases in Veneto were confirmed. B) On February 23, 2020 (day 3), evidence for local transmission from "patient one" increased and new cases of infections was discovered in the municipality of Vo' (Province of Padua). Ten municipalities in Lombardy and one in the Providence of Padua, identified as hotspots, were maintained under strict lockdown (i.e.,., labeled as critical red areas), while some preventive restrictions (e.g.,., temporary closure of schools and universities) were enforced in some regions. C) On March 8, 2020 (day 17), the whole of Lombardy and 14 Italian provinces (including the Province of Padua) were set under lockdown by the application of the Prime Ministerial Decree (DPCM) of August 03, 2020. Social distancing measures were implemented in the whole country. D) On March 10, 2020 (day 19), the lockdown area was extended to the whole nation by the application of the Prime Ministerial Decree (DPCM) of March 09, 2020; progressive restrictive limitations on mobility and social distance were also instituted.

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We express our kind appreciation to A. Corazzina, E. Ravazzolo and M. Riondato, support staff of the LIMA Laboratory (University of Padua, DCTV) for their precious technical assistance.