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 million 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 reverse transcriptase quantitative PCR (RT-qPCR) assays, targeting the polymerase (IP2 and IP4), the envelope (E), and the nucleoprotein (N1 and N2) genome regions, were employed for severe acute respiratory syndrome coronavirus 2 (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 (WBE) 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 the 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 to 6.5% was figured. On the other hand, proportions of around 0.12% and 0.09% of the total population were 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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 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 by 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. Health authorities profited from WBE to complement other inputs and adopt rapid and adequate measures to mitigate the effects of the pandemic. For example, sentinel surveillance of specific sewers helped to locate COVID-19 hot spots and to conduct massive numbers of RT-PCR tests among the population.

Keywords: COVID-19; SARS-CoV-2; early warning; epidemiology; sewage; surveillance.

FIG 1

Evolution of SARS-CoV-2 genomes in two large Barcelona wastewater treatment plants (WWTP). (A and D) Detection of the RNA-dependent RNA polymerase gene (IP2 and IP4 primers). (B and E) Detection of the envelope protein gene (E primers). (C and F) Detection of the nucleoprotein gene (N1 primers). The absence of values on a given date is due to the unavailability of aliquots to assay. Dashed lines depict limits of detection. Red, orange, and green arrows indicate phase 1, phase 2, and phase 3 of the deconfinement, respectively (Table S1).

FIG 2

Cumulated SARS-CoV-2 shedders associated with WWTP1 and WWTP2, determined by estimating fecal excretion periods of 7, 14, and 21 days and based on the actual number of reported symptomatic cases.

FIG 3

SARS-CoV-2 genome copy levels in grab samples from four urban sewers, detected with primers for IP2, IP4, E, N1, and N2. Sewer1 drains into WWTP1, while sewer2, sewer3, and sewer4 drain into WWTP2.

FIG 4

Estimation of the total number of SARS-CoV-2 infected shedders, including symptomatic, presymptomatic, asymptomatic, and undiagnosed cases. A model was developed based on the genome copies at the wastewater treatment plants detected during the first wave of the pandemic using the E target; the reported numbers of genome copies excreted in feces were also determined by employing the E target (11) and considering the actual daily wastewater flow.