Time Evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Wastewater during the First Pandemic Wave of COVID-19 in the Metropolitan Area of Barcelona, Spain

Abstract

Two large wastewater treatment plants (WWTP), covering around 2.7 M inhabitants, which represents around 85% of the metropolitan area of Barcelona, were sampled before, during and after the implementation of a complete lockdown. Five one-step RT-qPCR assays, targeting the polymerase (IP2 and IP4), the envelope E and the nucleoprotein (N1 and N2) genome regions, were employed for SARS-CoV-2 RNA detection in 24-h composite wastewater samples concentrated by polyethylene glycol (PEG) precipitation. SARS-CoV-2 was detected in a sewage sample collected 41 days ahead of the declaration of the first COVID-19 case. The evolution of SARS-CoV-2 genome copies in wastewater evidenced the validity of water-based epidemiology to anticipate COVID-19 outbreaks, to evaluate the impact of control measures and even to estimate the burden of shedders, including presymptomatic, asymptomatic, symptomatic and undiagnosed cases. For this latter objective, a model was applied for the estimation of the total number of shedders, evidencing a high proportion of asymptomatic infected individuals. In this way, an infection prevalence of 2.0-6.5% was figured. On the other hand, a proportion of around 0.12% and 0.09% of the total population was determined to be required for positive detection in the two WWTPs. At the end of the lockdown, SARS-CoV-2 RNA apparently disappeared in the WWTPs but could still be detected in grab samples from four urban sewers. Sewer monitoring allowed for location of specific hot spots of COVID-19, enabling the rapid adoption of appropriate mitigation measures. IMPORTANCE Water-based epidemiology (WBE) is a valuable early warning tool for tracking the circulation of the virus among the population, including not only symptomatic patients, but also asymptomatic, presymptomatic and misdiagnosed carriers, which represent a high proportion of the infected population. In the specific case of Barcelona, wastewater surveillance anticipated several weeks not only the original COVID-19 pandemic wave, but also the onset of the second wave. In addition, SARS-CoV-2 occurrence in wastewater evidenced the efficacy of the adopted lockdown measures on the circulation of the virus.

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

“…The US CDC N1 assay also outperformed the Charité assay and assays targeting the M gene and IP2/IP4 (RdRP) regions when applied to quantification of SARS-CoV-2 from wastewater in Spain (Perez-Cataluna et al, 2021). In another study, the US CDC N1 assay resulted in a longer period of positive detections in wastewater compared to IP2 and IP4, the E genome regions, while the US CDC N2 assay provided inconsistent results (Chavarria-Miro et al, 2021). Fig.…”

“…Intact or degraded SARS-CoV-2 viruses and RNA fragments reach sewer networks through toilets, showers, wash basins and sinks and can subsequently be detected in wastewater, both at the sub-catchment scale and in the influent to wastewater treatment plants (WWTPs). Recent studies have highlighted the potential application of SARS-CoV-2 RNA surveillance in untreated wastewater could provide information on the early detection or act as an advance warning system for the circulation of COVID-19 in a community (Ahmed et al, 2020a;Ahmed et al, 2020b;La Rosa et al, 2020;Medema et al, 2020;Miyani et al, 2020;Randazzo et al, 2020;Rimoldi et al, 2020;Wu et al, 2020;Chavarria-Miro et al, 2021;Gerrity et al, 2021). A caveat common to all methods based solely on molecular detection is that wastewater surveillance measures the presence and quantity of SARS-CoV-2 RNA, not the viable or infectious viruses from the individual building level to large metropolitan areas (i.e., WWTP influents).…”

Enumerating asymptomatic COVID-19 cases and estimating SARS-CoV-2 fecal shedding rates via wastewater-based epidemiology

“…The fecal shedding rate of SARS-CoV-2 RNA per gram of feces from an infected individual was enumerated based on known concentrations of viral RNA in positive wastewater samples and the fact that the total numbers of infected persons contributing to the total virus load in the samples were also known. Equations from previous reports using viral loads in wastewater to estimate the number of infected persons in a community (Ahmed et al, 2020;Chavarria-Miró et al, 2021;Curtis et al, 2020) were modified so that the fecal shedding rate of viral RNA per infected person could be quantified. The fecal shedding rate (FS) in terms of genome copies per gram-feces (gc/g-feces) was calculated as follows:…”

“…Fecal shedding rates of SARS-CoV-2 RNA were estimated from dorm wastewater samples using a modified equation from previous reports (Ahmed et al, 2020;Chavarria-Miró et al, 2021;Curtis et al, 2020). This calculation accounts for the number of infected people within a defined community, the amount of fecal material excreted, the shedding rate of infected individuals, the flow rate of the wastewater, and the viral load within the wastewater.…”

“…Shedding rates in this study are based on young university students who reside in dormitories and do not necessarily approximate the general population. Nevertheless, a recent study estimated the total number of active shedders from SARS-CoV-2 RNA levels in wastewater from a large metropolitan area similarly demonstrating that shedding resulted from a high proportion of asymptomatic individuals (Chavarria-Miró et al, 2021).…”