key: cord-320787-dwyyjq6o
authors: La Rosa, Giuseppina; Iaconelli, Marcello; Mancini, Pamela; Ferraro, Giusy Bonanno; Veneri, Carolina; Bonadonna, Lucia; Lucentini, Luca; Suffredini, Elisabetta
title: First detection of SARS-CoV-2 in untreated wastewaters in Italy
date: 2020-05-23
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.139652
sha: 
doc_id: 320787
cord_uid: dwyyjq6o

Abstract Several studies have demonstrated the advantages of environmental surveillance through the monitoring of sewage for the assessment of viruses circulating in a given community (wastewater-based epidemiology, WBE). During the COVID-19 public health emergency, many reports have described the presence of SARS-CoV-2 RNA in stools from COVID-19 patients, and a few studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide. Italy is among the world's worst-affected countries in the COVID-19 pandemic, but so far there are no studies assessing the presence of SARS-CoV-2 in Italian wastewaters. To this aim, twelve influent sewage samples, collected between February and April 2020 from Wastewater Treatment Plants in Milan and Rome, were tested adapting, for concentration, the standard WHO procedure for Poliovirus surveillance. Molecular analysis was undertaken with three nested protocols, including a newly designed SARS-CoV-2 specific primer set. SARS-CoV-2 RNA detection was accomplished in volumes of 250 mL of wastewaters collected in areas of high (Milan) and low (Rome) epidemic circulation, according to clinical data. Overall, 6 out of 12 samples were positive. One of the positive results was obtained in a Milan wastewater sample collected a few days after the first notified Italian case of autochthonous SARS-CoV-2. The study confirms that WBE has the potential to be applied to SARS-CoV-2 as a sensitive tool to study spatial and temporal trends of virus circulation in the population.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease COVID-19, a public health emergency worldwide. On March 11 th 2020, the World Health Organization declared COVID-19 a pandemic. Italy is among the world's most affected countries in the COVID-19 pandemic. Indeed, after entering Italy, COVID-19 has been spreading fast. As of April 20 th 2020, the total number of cases reported by the authorities reached 181,228, with 108,237 active cases (Dipartimento della Protezione Civile, Bulletin 20.04.2020), mainly located in Northern Italy (Lombardy, and its neighbouring regions of Emilia-Romagna and Piedmont).

Presymptomatic and paucisymptomatic carriers, mostly undetected in clinical and laboratory surveillance systems, contribute to the spread of the disease (Bay et al., 2020; Nicastri et al., 2020; Rothe et al., 2020; WHO, 2020) and hamper the efforts made to assess the extent of SARS-CoV-2 circulation in the population and to control efficiently virus transmission. Analytical regular investigation of wastewaters provides valuable information to measure viral circulation in the population as Wastewater Treatment Plants (WWTPs), collecting and concentrating human excreta, are useful sampling points receiving discharges from the entire community.

Environmental microbiologists have studied pathogens in sewage for decades (La Rosa & Muscillo, 2012; Sinclair et al., 2008) . The screening of wastewater, as a public health surveillance tool, defined as wastewater-based epidemiology (WBE), is currently well recognized (Daughton, 2018; Xagoraraki & O'Brien, 2020) . In the recent years, scientists have applied WBE to a wide range of waterborne, foodborne and fecal-oral viruses, which infected individuals usually excrete in high concentration with faeces (Katayama et al., 2008; Iaconelli et al., 2017; Bisseaux et al., 2018) .

However, the concept of WBE can also be applied to viruses beyond those commonly associated with the faecal-oral route (i.e. enteric viruses), since viral shedding may involve different body fluids ultimately discharged into urban sewage.

Some studies have reported the presence of viral RNA in the stools of COVID-19 patients in percentages ranging from 16.5% to 100% at a concentration up to 6.8 log 10 genome copies/g of stool (Chen et al., 200; Lo et al., 2000; Han et al., 2000; Lescure et al., 2000) . Furthermore, preliminary studies have reported the detection of SARS-CoV-2 RNA in wastewater in The Netherlands (Medema et al., 2020) , France (Wurtzer et al., 2020) , USA (Wu et al., 2020), and Australia (Ashmed et al., 2020) . To date, no study has yet provided insights into the presence of SARS-CoV-2 in wastewaters in Italy.

Herein we report the results of the screening for SARS-CoV-2 presence in sewage samples collected between the end of February and the beginning of April 2020 from WWTPs in Milan (Northern Italy) and Rome (Central Italy).

Twelve raw sewage samples were collected between the 3 rd of February and the 2 nd of April 2020 from three WWTPs, located in Milan (two distinct plants, reported as A and B) and in Rome (one plant receiving two different pipelines, C1 and C2, from different districts of the town), respectively. Total numbers of inhabitants served by these WWTPs (expressed as population equivalents) were 1.050.000, 1.050.000, and 900.000, for Plant A, B, and C, respectively.

Composite samples, representing 24-hour period were collected from the WWTP influent, immediately stored at -20 °C, and dispatched frozen to the National Institute of Health for analysis.

Before viral concentration, samples underwent a 30 min treatment at 56°C to increase the safety of the analytical protocol for the laboratory personnel and environment. After heat treatment, samples were processed using Class II biological safety cabinets, and standard precautions were applied J o u r n a l P r e -p r o o f (hand hygiene products and personal protective equipment e.g., gloves, gowns, face and eye protection).

Sample concentration took place using a two-phase (PEG-dextran method) separation as detailed in the 2003 WHO Guidelines for Environmental Surveillance of Poliovirus protocol (WHO, 2003) , with modifications to adapt the protocol to enveloped viruses. In brief, the wastewater sample (250 mL) was centrifuged to pellet the wastewater solids, retaining the pellet for further processing. The clarified wastewater was mixed with dextran and polyethylene glycol (PEG), and the mixture was left to stand overnight at 4°C in a separation funnel. The bottom layer and the interphase were then collected drop-wise, and this concentrate was added to the pellet from the initial centrifugation. The chloroform treatment that the WHO protocol envisages at this stage was omitted to preserve the integrity of the enveloped viruses object of this study. The extraction of viral RNA was done using the NucliSENS miniMAG semi-automated extraction system with magnetic silica carried out following manufacturer's instructions (bioMerieux, Marcy l'Etoile, France) with however slight modifications. The lysis phase was prolonged to 20 minutes, and brief centrifugation (2000 × g, 1 min) was used to pellet the sediment; subsequently, magnetic silica beads were added to the cleaned supernatant. Before molecular tests, the extracted nucleic acids were further purified from potential PCR inhibitors using the OneStep PCR Inhibitor Removal Kit (Zymo Research, CA, USA).

In the absence of a standardized method for SARS-CoV-2 detection in environmental samples, RNAs were tested for the presence of SARS-CoV-2 using three different nested RT-PCR assays and one real-time qPCR assay (Table 1 and Figure 1 b) a newly designed primer set specific for SARS-CoV-2. Novel nested primers, amplifying a 332 bp fragment of ORF1ab, were designed using Primer3 software (http://primer3.ut.ee/).

For the assays a) and b) first-strand cDNA was synthesized using Super Script IV Reverse Transcriptase (ThermoFisher Scientific) with the reverse primer. PCR reaction was performed using 2.5 µl of cDNA in a final volume of 25 µl (Kit Platinum™ SuperFi™ Green PCR Master Mix, Thermo), using 1 µl of primers (10 µM). The PCR conditions were as follows: 98 °C for 30 sec; 35 cycles of 98 °C for 10 sec, 50 °C and 54 °C for 10 sec for assay a) and b), respectively, and 72 °C for 30 sec; final extension 72 °C for 5 min. After the first round PCR, nested PCR was performed using 2 µl of first PCR product and under the same reaction composition and thermal profile conditions. A synthetic DNA (Biofab Research, Italy) including the PCR target region, was used to set up PCR conditions before experiments with study samples, but was not amplified along with samples to avoid risks of PCR contamination. Molecular grade water was used as the negative control. c) a published nested RT-PCR for SARS-CoV-2 targeting the spike region (Nao et al., 2020). cDNA was synthesized from 5 l of sample RNA, using SuperScript III Reverse Transcriptase (ThermoFisher Scientific), 0.5 M of the reverse primer (WuhanCoV-spk2-r, Table 1 ) and a 50 min reaction at 50 °C (20 l final volume). First PCR reaction was performed by adding the reaction mix (Dream Taq polymerase and buffer from ThermoFisher Scientific, 0.4 M of primers WuhanCoV-spk2-r and WuhanCoV-spk1-f) directly to the whole volume of synthesized cDNA. The used PCR conditions were as follows: 95°C for 1 min; 35 cycles of 95 °C for 30 sec, 56 °C for 30 sec, and 72 °C for 40 sec; final extension 72 °C for 5 min. Nested PCR (primers NIID_WH-1_F24381 and NIID_WH-1_R24873) was performed in a total volume of 50 µl using 5 µl of first PCR product, with the same conditions applied for the first PCR and 45 cycles. All reactions were performed in duplicate. For standard curve construction, the targeted region, coupled with a T7 promoter, was synthetized and quantified by Eurofins Genomics (Germany), and tenfold dilutions were used for curve construction. In vitro synthetized RNA using the standard curve DNA as a template was used as an external amplification control to check for PCR inhibition. All amplifications were conducted on a QuantStudio 12K instrument (Thermo Scientific). Molecular biology water served as a non-template control.

All samples were retested for confirmation of results obtained with methods a), b), and c). The PCR products were revealed by electrophoresis on 2% agarose gels and were purified using a Montage PCRm96 Microwell Filter Plate (Millipore, Billerica, MA, USA) and then direct sequenced on both strands (BioFab Research, Rome, Italy). Sequences were identified in terms of the closest homology sequence using BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi. All Italian SARS-CoV-2 genome sequences available at the time of analysis were retrieved from Gisaid (https://www.gisaid.org/) for comparison with study sequences, using the MEGA X software (Kumar et al., 2018) ..

Sequences were submitted to NCBI GenBank with the accession numbers: MT373156-MT373163.

The 50% (6/12) of the wastewater samples showed positive results for SARS-CoV-2 RNA, and the newly designed assay in the RdRp gene showed a higher sensitivity compared to the assay targeting J o u r n a l P r e -p r o o f the spike gene (Table 2 ). Both the published and newly designed SARS-CoV-2 specific primer sets detected bands of the expected size and were confirmed by sequencing. In contrast, only unspecific products were detected with a broad range assay for coronavirus. Upon comparison of broad range primers with SARS-CoV-2 genome, we noted that they showed only 77.1 to 91.3 % nt identity, which explain why these were not able to amplify the novel coronavirus. No positive results were obtained by real-time RT-qPCR, therefore no quantitative data could be provided for the positive samples. This may be related to the sensitivity of the RdRp assay used in this study. Indeed, in recent comparative studies, the sensitivity of this assay was shown to be low compared to others developed by WHO referral laboratories (Etievant, 2020). In particular, the limit of detection (LOD) of this assay was estimated at 316 viral genomic equivalents per reaction by Nalla and coworkers (2020) and above 500 genome copies per reaction by Vogel et al. (2020) as well as in our hands (data not shown), suggesting that virus concentration was below the LOD of the assay. However, the external inhibition control associated to this assay was useful to confirm the acceptable levels of PCR inhibitors, all samples being below the acceptability criterion (median inhibition 29.1%, range 8.7%-51.4%).

In this study, a thermal treatment of samples (30 min at 56 °C) was included before concentration to increase the safety for the laboratory personnel during sample manipulation. These conditions were reported to reduce the virulence of SARS-CoV-2 by over 5 log without affecting RNA integrity Following this investigation on the occurrence of SARS-CoV-2 RNA in sewage, the production of quantitative data on virus concentration in raw sewage will be undertaken with the use of molecular methods optimized for environmental samples. This approach will allow obtaining a rough estimation of the total number of subjects excreting the virus, by integrating -as done by Wu and co-workers in samples taken in the United States (Wu et al., 2020) -the available information on viral shedding rates, WWTPs loads, and virus concentration in wastewaters. Moreover, the environmental surveillance will be extended to the collection of wastewater samples available in the Department of Environment and Health of the Italian National Health Institute, that were collected throughout Italy in the framework of different projects on enteric viruses. Such monitoring will provide a picture of the SARS-CoV-2 circulation across the different regions of Italy and over time, to better understand the virus circulation, as provided by wastewater-based epidemiology (WBE) and compare it to the clinical data. Samples collected before the reporting of the first known Italian case on February 21 will also be tested, to possibly infer when SARS-CoV-2 first appeared in Italy.

In a previous study, indeed, wastewater monitoring provided evidence that a novel variant of Norovirus GII.17 (termed Kawasaki 2014) had been circulating in the Italian population before its first appearance and identification in clinical cases, later becoming one of the prevalent variants in the population (Suffredini et al., 2018) .

Also and most important, environmental monitoring of SARS-CoV-2 in sewage will continue when the emergency phase will be over, and its circulation in the population will be considered limited.

Indeed, sewage surveillance could also serve for the early detection of a possible re-emergence of COVID-19 in urban areas. WHO recommends environmental surveillance for poliovirus as an early Environmental monitoring, therefore, appears to be an effective measure for proving early warning against pathogen reintroduction.

In conclusions, the main findings of this study are: 1) first detection of SARS-CoV-2 RNA fragments in sewage in Italy;

2) demonstration of the suitability of the WHO protocol for sewage treatment to enveloped viruses after appropriate modifications;

3) design of a novel nested PCR assay specific for SARS-CoV-2, useful for screening purposes.

Further research will clarify the applicability of WBE to SARS-CoV-2 for prompt detection, the J o u r n a l P r e -p r o o f × SARS-CoV-2 not detected; ○ SARS-CoV-2 detected (ORF1ab); • SARS-CoV-2 detected (spike) a weak positive J o u r n a l P r e -p r o o f AUTHORSHIP STATEMENT All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Hong Kong Journal of Occupational Therapy. Authorship contributions Please indicate the specific contributions made by each author (list the authors' initials followed by their surnames, e.g., Y.L. Cheung). The name of each author must appear at least once in each of the three categories below.

Category 1 Conception and design of study: GLR, ES, LL, LB; acquisition of data: MI,PM, GBF, CV; analysis and/or interpretation of data: MI,PM, GBF, CV; GLR, ES, LB, LL Category 2 Drafting the manuscript: GLR, ES revising the manuscript critically for important intellectual content: GLR, ES Category 3 Approval of the version of the manuscript to be published (the names of all authors must be

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