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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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Letter to the Editor
SARS-Cov-2 RNA Found on Particulate Matter of Bergamo
in Northern Italy: First Preliminary Evidence
Leonardo Setti1, Fabrizio Passarini2, Gianluigi De Gennaro3, Pierluigi Barbieri4, Maria Grazia Perrone5,
Massimo Borelli6, Jolanda Palmisani3, Alessia Di Gilio3, Valentina Torboli6, Alberto Pallavicini6, Maurizio
Ruscio7, Prisco Piscitelli8, Alessandro Miani8
1. Dept. Industrial Chemistry, University of Bologna, Viale del Risorgimento – 4, I-40136, Bologna, Italy
e-mail: [email protected]
2. Interdepartmental Centre for Industrial Research "Renewable Sources, Environment, Blue Growth, Energy",
University of Bologna, Rimini, Italy e-mail: [email protected]
3. Dept. of Biology, University “Aldo Moro” of Bari, Bari, Italy
e-mail: [email protected]; [email protected]; [email protected]
4. Dept. of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy
e-mail: [email protected]
5. Environmental Research Division, TCR TECORA, Milan, Italy
e-mail: [email protected]
6. Dept. of Life Sciences - University of Trieste, Trieste, Italy
e-mail: [email protected]; [email protected]; [email protected]
7. Division of Laboratory Medicine, University Hospital Giuliano Isontina (ASU GI), Trieste, Italy
email: [email protected]
8. Italian Society of Environmental Medicine (SIMA), Milan, Italy
e-mail: [email protected]; [email protected]
Corresponding Author:
Leonardo Setti, Department of Industrial Chemistry, University of Bologna
Viale del Risorgimento 4, 40136, Bologna, Italy; e-mail: [email protected]
Severe acute respiratory syndrome known as COVID-19 disease - due to SARS-CoV-2 virus - is
recognized to spread via respiratory droplets and close contacts.[1] The burden of COVID-19 was
extremely severe in Lombardy and Po Valley (Northern Italy),[2] an area characterized by high
concentrations of particulate matter, which are already known to have negative effects on human
health.[3] Regional figures are available for Italy at the date of April 12th show that about 30% of
currently positive people still live in Lombardy (about 40% if considering the overall cases confirmed
from the beginning of the epidemic), followed by Emilia Romagna (13.5% of currently positive people),
Piedmont (10.5%), and Veneto (10%).[2] These four regions of the Po Valley account for 80% of total
deaths recorded in Italy and 65% of Intensive Care Units admissions.[2] A research carried out by the
Harvard School of Public Health seems to confirm an association between increases in PM
concentrations and mortality rates due to COVID-19.[4]
medRxiv preprint doi: https://doi.org/10.1101/2020.04.15.20065995.this version posted April 24, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
In previous communications, we have hypothesized the possibility that SARS-CoV-2 virus could be
present on particulate matter (PM) during the spreading of the infection, [5,6] consistently with evidence
already available for other viruses [7-15]. However, the issue of airborne PM-associated microbiome,
especially in urban environments, remains largely under-investigated [16], and – at the present –
nobody has still carried out experimental studies specifically aimed at confirming or excluding the
presence of the SARS-CoV-2 on PM.
Here, we present the first results of the analyses that we have performed on 34 PM10 samples of
outdoor/airborne PM10 from an industrial site of Bergamo Province, collected with two air samplers
(POS1 and POS2) over a continuous 3-weeks period, from February 21st to March 13th.
Following the methodology described by Pan et al. in 2019 [17] for the collection, particle sizing and
detection of airborne viruses, PM samples were collected on quartz fiber filters by using a low-volume
gravimetric air sampler (38.3 l/min for 23 h), compliant with the reference method EN12341:2014 for
PM10 monitoring. Particulate matter was trapped onto filters with 99.9% typical aerosol retention,
properly stored and delivered to the laboratory of Applied and Comparative Genomics of Trieste
University. Given the "environmental" nature of the sample, presumably rich in inhibitors of DNA
polymerases, we proceeded with the extraction of RNA by using the Quick RNA fecal soil microbe kit
adapted to the type of the filters.[18] 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 ul of solution, which was then processed as defined by the standard
protocols, resulting in a final eluate of 15 ul. Subsequently, 5 ul were used for the SARS-CoV-2 testing.
Given the particular origin of the sample, the qScript XLT 1-Step RT-qPCR ToughMix was used.[19]
The amplification systems were those of the protocol developed by Corman et al, published on the
WHO website [20].
The test was explicitly aimed at confirming or excluding the presence of the SARS-CoV-2 RNA on
particulate matter. The first analysis used the “E gene” as a molecular marker and produced an
impressive positive result on 15 out of 16 filters even if, as we could expect, the Ct was between 36-38
cycles. After that, we have replicated the analysis on 6 of the positive filters (already positive to “E
gene”) by using the “RtDR gene” as a molecular marker – which is highly specific for SARS-CoV-2 –
reaching 5 significant results of positivity; control tests to exclude false positivity were also successfully
medRxiv preprint doi: https://doi.org/10.1101/2020.04.15.20065995.this version posted April 24, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
performed (Fig. 1).
To avoid the running out of the scarce sampling material available, the remaining extracted RNAs were
delivered to the local University Hospital (one of the clinical centres authorized by the Italian
Government for SARS-CoV-2 diagnostic tests), in order to perform a second parallel blind test. This
second clinical laboratory tested 34 RNA extractions for the E, N and RdRP genes, reporting 7 positive
results for at least one of the three marker genes, with positivity separately confirmed for all the three
markers (Tab. 1). Because of the nature of the sample, and considering that the sampling has not been
carried out for clinical diagnostic purposes but for environmental pollution tests (taking also into
account that filters were stored for at least four weeks before undergoing molecular genetic analyses, as
a consequence of the Italian shutdown), we can confirm to have reasonably demonstrated the presence
of SARS-CoV-2 viral RNA by detecting highly specific “RtDR gene” on 8 filters. However, due to the
lack of additional materials from the filters, we were not able to repeat enough number of tests to show
positivity for all the 3 molecular markers simultaneously.
This is the first preliminary evidence that SARS-CoV-2 RNA can be present on outdoor particulate
matter, thus suggesting that, in conditions of atmospheric stability and high concentrations of PM,
SARS-CoV-2 could create clusters with outdoor PM and – by reducing their diffusion coefficient –
enhance the persistence of the virus in the atmosphere. Further confirmations of this preliminary
evidence are ongoing, and should include real-time assessment about the vitality of the SARS-CoV-2 as
well as its virulence when adsorbed on particulate matter. At the present, no assumptions can be made
concerning the presence of the virus on PM and COVID-19 outbreak progression. Other issues to be
specifically addressed are the average concentrations of PM eventually required for a potential “boost
effect” of the contagion in the areas experiencing the most dramatic burden of COVID-19, or even the
theoretic possibility of immunization consequent to minimal dose exposures at lower thresholds of PM.
medRxiv preprint doi: https://doi.org/10.1101/2020.04.15.20065995.this version posted April 24, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
Fig.1 Amplification curves of E (A) and RdRP genes (B): green lines represent tested filters; cross line
lin
represents reference filter extractions; red lines represent the amplification of the positive samples.
Fig.2. Positive results (marked with X) for E, N and RdRP genes obtained for all the PM10 sample
ples
tested by the two parallel genetic analyses.
medRxiv preprint doi: https://doi.org/10.1101/2020.04.15.20065995.this version posted April 24, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
References
1. World Health Organisation, Modes of transmission of virus causing COVID-19: implications for IPC
precaution recommendations, Scientific brief; available at: https://www.who.int/news-
room/commentaries/detail/modes-of-transmission-of-virus-causing-covid-19-implications-for-ipc-
precaution-recommendations (29 March 2020)
2. Italian Ministry of Health, daily bulletin Covid-19 outbreak in Italy, available at
http://www.salute.gov.it/imgs/C_17_notizie_4451_0_file.pdf
3. EEA, European Environmental Agency, Air Quality in Europe 2019 Report; No 10/2019; European
Environment Agency: Copenhagen, Denmark, availbale at: https://www.eea.europa.eu/publications/air-
quality-in-europe-2019
4. Xiao Wu, Rachel C. Nethery, M. Benjamin Sabath, Danielle Braun, Francesca Dominici, Exposure to air
pollution and COVID-19 mortality in the United States, available at:
https://projects.iq.harvard.edu/files/covid-pm/files/pm_and_covid_mortality.pdf
5. Italian Society of Environmental Medicine (SIMA), Position Paper Particulate Matter and COVID-19,
available at: http://www.simaonlus.it/wpsima/wp-content/uploads/2020/03/COVID_19_position-
paper_ENG.pdf
6. Setti L., Passarini F., De Gennaro G., Barbieri P., Perrone M.G., Piazzalunga A., Borelli M., Palmisani J.,
Di Gilio A, Piscitelli P, Miani A., Is there a Plausible Role for Particulate Matter in the spreading of
COVID-19 in Northern Italy?, BMJ Rapid Responses, April 8th 2020, available at:
https://www.bmj.com/content/368/bmj.m1103/rapid-responses
7. Sedlmaier, N., Hoppenheidt, K., Krist, H., Lehmann, S., Lang, H., Buttner, M. Generation of avian
influenza virus (AIV) contaminated fecal fine particulate matter (PM2.5): genome and infectivity detection
and calculation of immission. Veterinary Microbiology. 139, 156-164 (2009)
8. Zhao, Y., Richardson, B., Takle, E., Chai, L., Schmitt, D., Win, H. Airborne transmission may have
played a role in the spread of 2015 highly pathogenic avian influenza outbreaks in the United States. Sci
Rep. 9, 11755. https://doi.org/10.1038/s41598-019-47788-z (2019)
9. Ma, Y., Zhou, J., Yang, S., Zhao, Y., Zheng, X. Assessment for the impact of dust events on measles
incidence in western China. Atmospheric Environment. 157, 1-9 (2017)
10. Sorensen, J. H., Mackay, D. K. J., Jensen, C. Ø., Donaldson, A. I. An integrated model to predict the
atmospheric spread of foot-and-mouth disease virus Epidemiol. Infect., 124, 577–590 (2000)
11. Glostera, J., Alexandersen, S. New Directions: Airborne Transmission of Foot-and-Mouth Disease Virus
Atmospheric Environment, 38 (3), 503-505 (2004)
12. Reche, I., D’Orta, G., Mladenov, N., Winget, D.M., Suttle, C.A. Deposition rates of viruses and bacteria
above the atmosperic boundary layer. The ISME Journal. 12, 1154-1162 (2018)
13. Qin, N., Liang, P., Wu, C., Wang, G., Xu, Q., Xiong, X., Wang, T., Zolfo, M., Segata, N., Qin, H.,
Knight, R., Gilbert, J.A., Zhu, T.F. Longitudinal survey of microbiome associated with particulate matter
in a megacity. Genome Biology. 21, 55 (2020)
14. Zhao, Y., Richardson, B., Takle, E., Chai, L., Schmitt, D., Win, H. Airborne transmission may have
played a role in the spread of 2015 highly pathogenic avian influenza outbreaks in the United States. Sci
Rep. 9, 11755. https://doi.org/10.1038/s41598-019-47788-z (2019)
15. Ma, Y., Zhou, J., Yang, S., Zhao, Y., Zheng, X. Assessment for the impact of dust events on measles
incidence in western China. Atmospheric Environment. 157, 1-9 (2017)
16. Jiang, W., Laing, P., Wang, B., Fang, J.,Lang, J., Tian, G., Jiang, J., Zhu, T.F. Optimized DNA extraction
and metagenomic sequencing of airborne microbial communities. Nat. Protoc. 10, 768-779 (2015)
17. Pan, M., Lednicky, J.A., Wu, C.-Y., Collection, particle sizing and detection of airborne viruses. Journal
of Applied Microbiology, 127, 1596-1611 (2019)
18. Zymoresearch Ldt, product description, available at: https://www.zymoresearch.com/products/quick-rna-
fecal-soil-microbe-microprep-kit
19. Quantabio Ltd, descriprion of the product, available at: https://www.quantabio.com/qscript-xlt-1-step-
rt-qpcr-toughmix
20. Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D. K., & Mulders, D. G. (2020).
Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance, 25(3),
available at:.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988269/
