key: cord-1038782-lku64i31
authors: Kayalar, O.; Ari, A.; Babuscu, G.; Konyalilar, N.; Dogan, O.; Can, F.; Sahin, U. A.; Gaga, E.; Kuzu, L.; Ari, P.; Odabasi, M.; Tasdemir, Y.; Cindoruk, S.; Esen, F.; Sakin, E.; Caliskan, B.; Tecer, L.; Ficici, M.; Altin, A.; Onat, B.; Ayvaz, C.; Uzun, B.; Saral, A.; Dogeroglu, T.; Malkoc, S.; Uzmez, O.; Kunt, F.; Aydin, S.; Kara, M.; Yaman, B.; Dogan, G.; Olgun, B.; Dokumaci, E.; Gullu, G.; Uzunpinar, E.; Bayram, H.
title: Existence of SARS-CoV-2 RNA on ambient particulate matter samples: A nationwide study in Turkey
date: 2021-01-25
journal: nan
DOI: 10.1101/2021.01.24.21250391
sha: c61847adc7c241b4dda132925f80b94696833d1c
doc_id: 1038782
cord_uid: lku64i31

Coronavirus disease 2019 (COVID-19) is caused by the SARS-CoV-2 virus and has been affecting the world since the end of 2019. Turkey is severely affected with the first case being reported on March 11th 2020. Ambient particulate matter (PM) samples in various size ranges were collected from 13 sites including urban and urban background locations and hospital gardens in 10 cities across Turkey between the 13th of May and the 14th of June, 2020 to investigate a possible presence of SARS-CoV-2 RNA on ambient PM. A total of 155 daily samples (TSP, n=80; PM2.5, n=33; PM2.5-10, n=23; PM10, n=19; and 6 size segregated, n=48) were collected using various samplers in each city. The N1 gene and RdRP gene expressions were analyzed for the presence of SARS-CoV-2 as suggested by the Centers for Disease Control and Prevention (CDC). According to RT-PCR and 3D-RT-PCR analysis, dual RdRP and N1 gene positivity were detected in 20 (9.8 %) of the samples. The highest percentage of virus detection on PM samples was from hospital gardens in Tekirdag, Zonguldak, and Istanbul, especially in PM2.5 mode. Samples collected from two urban sites were also positive. Findings of this study have suggested that SARS CoV2 may be transported by ambient particles especially at sites close to the infection hot-spots. However, whether this has an impact on the spread of the virus infection remains to be determined.

After the first reported cases of unknown pneumonia in Wuhan, China, in December 2019, the World Health Organization (WHO) announced an International Public Health Emergency in January and a pandemic in March 2020. A novel and previously unknown severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) was isolated from the epithelial cells of the patients with pneumonia. This was later named Coronavirus by the WHO in February 2020 (1) (2) (3) . The spread of the outbreak continues (4) . According to WHO, the COVID-19 pandemic has resulted in 47,596,852 confirmed cases and 1,216,357 related deaths globally as of November 05, 2020; the number of cases and deaths continues to increase (2) .

Accordingly, the total number of cases and the number of deaths were 384,509 and 10,558, respectively, in Turkey (5) .

Ambient and indoor particulate matter (PM) is a complex matrix that may contain various chemical and biological constituents (bacteria, virus, and fungi etc.) of a great health concern (6-15). Medium-and long-range transmission of bacteria and virus species on the atmospheric PM have been recently studied (11, (16) (17) (18) (19) (20) . Accordingly, aerosol and droplets generated during speaking, sneezing, or coughing by infected people are well-known as the source of short-range transmission pathways for viral infections (21, 22) . In particular, respiratory viral diseases can spread directly or indirectly through a virus-containing particle (droplet) among humans (23) (24) (25) (26) .

The primary transmission mode of COVID-19 is person-to-person contact through respiratory droplets generated by breathing, sneezing, coughing, and contact with an infected subject. Indirect contact with contaminated surfaces also transfers the virus to the mouth, nose, and eyes (27, 28) . Transmission can also be through inhalation of the exhaled virus in respiratory droplets because of the long-term survival of coronaviruses outside of its host organism (29, 30). According to their results, higher emission rates were achieved by an asymptomatic COVID-19 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint subject during both light and heavy exercise conditions as speaking or oral breathing whereas the symptomatic subject in resting conditions mostly has low emission rates. Similarly, Liu et al. (1) investigated the aerodynamic nature of SARS-CoV-2 in size-segregated indoor aerosol samples collected from different divisions of hospitals in Wuhan, China. They found very low SARS-CoV-2 RNA concentrations in isolation wards and aerated patient rooms with higher concentrations in patients' toilets. They also reported that the size distribution of the SARS-CoV-2 peaked on the smallest particles suggesting that the long-range transmission was a possible spreading route The mode of transmission of the infection must be precisely determined to curtail the pandemic.

In this study, we collected ambient PM samples in various size ranges from 13 sites in ten Turkish cities including urban and suburban locations as well as hospital gardens from May 13 to June 14, 2020. We demonstrated the presence of SARS-CoV-2 on PM collected from the various locations studied.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint

A total of 155 samples including TSP (n = 80), PM 2.5 (n = 33), PM 2.5-10 (n = 23), and PM 10 (n = 19) were collected. Additionally, 48 PM samples segregated in 6 sizes were collected during the 8 days of sampling in urban İ stanbul. Table 1 shows the positive results of the RdRP and N1 genes on filters from various sites inspected within this study. The virus presence together with environmental parameters of all samples collected at each location are given in Table S1 .

The positivity of the SARS-CoV-2 on aerosol samples was characterized by a multi-parameter decision approach. Figure 1 demonstrates the protocol for analysis of PM samples. Initially, the N1 gene was studied on all 203 PM samples including segregated ones by RT-PCR using Syber Green method. Of these, four samples were positive for the N1 gene with a Ct < 40 whereas 199 samples had a Ct value of ≥ 40 for the same gene. The latter were further analyzed for specific products by checking melting curves as well as specific products in a 2% agarose gel. Traces of specific products were detected in 52 samples, and these were further analyzed for RdRP genes together with four samples positive for the N1 gene by RT-PCR using the Taqman hybridization probe. Of these 56 samples, 4 samples had a Ct <40 for both N1 and RdRP genes, 33 samples had a Ct of ≥ 40 for N1 and Ct <40 for RdRP genes, and two samples had a Ct≥40 for both N1 and RdRP genes with a specific product in the gel for N1 gene. In total, 39 out of 56 samples were analyzed by 3D-dPCR for N1 gene, and 20 samples were positive for the N1 gene. The remaining 17 of 56 samples had a Ct≥40 for both N1 and RdRP genes with no sign of a specific product, and these samples were treated as negative for SARS-CoV-2. These 20 samples were amplified above 10 copies µL -1 and considered positive. Among the positive samples, the lowest copy number was 80 while the highest copy number was 504 copies on the filters (Table 1) Forty-eight PM samples were collected by an impactor having different size ranges (0. The virus was detected in size-segregated PM samples collected on three different days out of 8.

On two succeeding days, the virus was detected on particles >7.2 µm and on <0.49 µm and >7.2 µm for 14 th and 15 th of May, respectively. Due to the lower percentage and the random results of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint positive observations in size-segregated samples, we could not observe a systematic distribution according to PM sizes. Thus, a detailed description of the size distribution of SARS-CoV-2 in atmospheric particles needs more attention and systematic approaches for further research.

When background samples were analyzed neither the N1 nor the RdRP gene was amplified during the 45 cycles of QRT-PCR. Therefore, these samples were negative for the presence of viruses, and no interference was observed from the used filters and equipment. In the rest of 155 PM samples, positive counts of SARS-CoV-2 RNA on other ambient particles according to PM sizes were: PM 2.5 (n = 10), PM 10 (n = 1), and TSP (n = 4) ( Table 1) .

Of the samples positive for SARS-CoV-2, 13 samples were close to hospitals, and the 

We demonstrated that SARS-CoV-2 RNA can be present on ambient PM suggesting that this virus may be transported via PM pollution. Table S1 for each sample. It is clear from Table 1 and are smaller cities compared to others but have worse air pollution (45) . Tekirdağ is an industrialized city and Zonguldak is a coal mining area with coal-fired power plants.

Airborne transmission of SARS-CoV-2 was previously investigated by Bontempi (41) in the ambient atmosphere of Northern Italy in relation to the increasing concentrations of PM. After a basic correlation analysis of ambient PM data and confirmed cases obtained from the cities of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2021. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2021. transmission, the current study shows that the data on these parameters were not adequate to perform a statistical analysis. Nevertheless, our findings suggest that SARS-CoV-2 may be transported by ambient particles-whether this has an impact on the spread of the virus remains to be determined. Therefore, the public should use personal protection equipment such as face masks during outdoor activities. Future studies should focus on the viability and infectivity of the SARS-CoV-2 present on both indoor and ambient particles.

PM samples within various size-ranges were collected from 13 locations within 10 cities in western Turkey between 13 th May and 14 th June 2020 during the first peak of the outbreak in Turkey. Figure 3 shows the location map of PM sampling sites, and Table 2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint For all sampling locations, sampling started in the morning and finished at the same time on the following day. At the end of 24 hrs, each sample was placed into sterile petri dish using sterile equipment and stored at -20° C. Filters were cut into halves with a sterile scalpel: One half was immediately subjected to PCR analysis. The remaining halves of the filters were kept frozen for further chemical and bacteriological characterization. Since the main purpose of this study was to investigate the presence of SARS-CoV-2, filters were not weighed to avoid any contamination.

Field blanks (n=3) from each site were also analyzed together with samples to detect any contamination.

There are more than 300 air quality monitoring stations in Turkey operated by the Turkish Ministry Cycling conditions for Syber detection were as follows: in 5 min at 95° C, followed by 45 cycles of 10 sec at 95° C, 30 sec at 63° C, and 30 sec at 72°C, and final cooling step at 4° C. Cycling conditions for Taqman Hybridization was as follows: in 7 min at 95° C, followed by 45 cycles of 10 sec at 95° C, 1 min at 63° C, and 30 sec at 72° C followed by maintenance at 4° C. Gene sets were obtained from the Centers for Disease Control and Prevention (CDC). Primer sequences: 

The main difference of three dimensional (3D)-digital PCR (3D-PCR) from QRT-PCR is that the reaction volume is split over a high number of small partitions (from 500 up to millions) of a very small volume (currently from 6 nanoliters down to a few picolitres). After the PCR, each partition is scored either as positive or negative (binary or digital read-out). Statistical analysis of the results is then used to determine the absolute quantity of target DNA in a sample (68) . This method shows high sensitivity with the strategy of counting a single molecule. It also provides a high reliability and repeatability level. Furthermore, this method has been proven to work with high sensitivity in studies related to SARS-COV-2 (69).

Airborne PM may have an unknown interaction level with the virus resulting in a low or high copy number of the virus. Thus, we aimed to detect this interaction with highly sensitive-dPCR QuantStudio 3D TM System (Thermo, USA) in samples previously determined as positive or suspicious for the presence of RdRP and/or N1 genes (62, 69) . Therefore, we further analyzed these samples for the existence of the N1 gene, which is specific to SARS-CoV-2 using QuantStudio™ 3D Digital PCR Master Mix v2 and QuantStudio TM 3D 20K v2 chips according to the instructions of the manufacturer (Thermo, USA).

Here, 5 µL of cDNA was used for 3D-dPCR analyses. Gene and probe sets for N1 gene were obtained from the CDC as described above (66) . Primers used in real-time PCR analyses were also used in 3D-dPCR analyses. The probe sequence of N1 gene was FAM-5'-. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint 1 3 ACCCCGCATTACGTTTGGTGGACC-3'-BHQ1. Cycling conditions were as follows: in 10 min at 96° C, followed by 39 cycles of 30 sec at 63° C and 2 min at 98° C, and a final step of 63° C per 2 min followed by maintenance at 4° C. The chips were read in the QuantStudio 3D TM reader (Thermo, USA), and the results were interpreted in the dPCR AnalysisSuite TM app in the Thermo Fisher Connect TM Dashboard. Results with precision values ≤ 20% were selected to estimate the quantity of SARS-CoV-2 genomic copies based on 3D-dPCR (62).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 25, 2021. ; https://doi.org/10.1101/2021.01.24.21250391 doi: medRxiv preprint

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