Detection of SARS-CoV-2 in wastewater in Italy is described for the first time.• Use of the PEG/dextran concentration method for SARS-CoV-2 is reported. • A novel nested PCR assay specific for SARS-CoV-2 was designed. • Wastewater-based epidemiology can be applied for COVID-19 surveillance.
Several studies have demonstrated the advantages of environmental surveillance through the monitoring of sewer systems 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 occurred in volumes of 250 mL of wastewaters collected in both areas at 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 shows 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, and Italy is among the most severely affected countries. The first autochthonous Italian case of COVID-19 was documented on February 21, 2020. We investigated the possibility that SARS-CoV-2 emerged in Italy earlier than that date, by analysing 40 composite influent wastewater samples collected - in the framework of other wastewater-based epidemiology projects - between October 2019 and February 2020 from five wastewater treatment plants (WWTPs) in three cities and regions in northern Italy (Milan/Lombardy, Turin/Piedmont and Bologna/Emilia Romagna). Twenty-four additional samples collected in the same WWTPs between September 2018 and June 2019 (i.e. long before the onset of the epidemic) were included as ‘blank’ samples. Viral concentration was performed according to the standard World Health Organization procedure for poliovirus sewage surveillance, with modifications. Molecular analysis was undertaken with both nested RT-PCR and real-rime RT-PCR assays. A total of 15 positive samples were confirmed by both methods. The earliest dates back to 18 December 2019 in Milan and Turin and 29 January 2020 in Bologna. Virus concentration in the samples ranged from below the limit of detection (LOD) to 5.6 × 10
4
genome copies (g.c.)/L, and most of the samples (23 out of 26) were below the limit of quantification of PCR.
Our results demonstrate that SARS-CoV-2 was already circulating in northern Italy at the end of 2019. Moreover, it was circulating in different geographic regions simultaneously, which changes our previous understanding of the geographical circulation of the virus in Italy. Our study highlights the importance of environmental surveillance as an early warning system, to monitor the levels of virus circulating in the population and identify outbreaks even before cases are notified to the healthcare system.
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