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.

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.

Tracing surface and airborne SARS-CoV-2 RNA inside public buses and subway trains.

Given the widespread concern but general lack of information over the possibility of SARS-CoV-2 infection in public transport, key issues such as passenger personal hygiene, efficient air circulation systems, and the effective disinfection of frequently touched surfaces need to be evaluated to educate the public and diminish the risk of viral transmission as we learn to live with the ongoing pandemic. In this context we report on a study involving the collection of 99 samples taken from inside Barcelona buses and subway trains in May to July 2020. From this sample group 82 (58 surface swabs, 9 air conditioning (a/c) filters, 3 a/c dust, 12 ambient air) were selected to be analysed by RT-PCR for traces of the SARS-CoV-2 virus. Thirty of these selected samples showed evidence for one or more of 3 target RNA gene regions specific for this virus (IP2, IP4, E). Most (24) of these 30 samples showed positivity for only 1 of the 3 RNA targets, 4 samples yielded 2 targets, and 2 samples provided evidence for all 3 targets. RNA remnants were more common in surface swabs from support bars (23 out of 58) than in ambient air inside the vehicles (3 out of 12), with relatively higher concentrations of viral RNA fragments in buses rather than in trains. Whereas subway train a/c filters examined were all virus-free, 4 of the 9 bus a/c filter/dust samples yielded evidence for viral RNA. After nocturnal maintenance and cleaning most buses initially yielding positive results subsequently showed elimination of the RT-PCR signal, although signs of viral RNA remained in 4 of 13 initially positive samples. The presence of such remnant viral traces however does not demonstrate infectivity, which in the present study is considered unlikely given the fragmentary nature of the gene targets detected. Nevertheless, best practice demands that close attention to ventilation systems and regular vehicle disinfection in public transport worldwide need to be rigorously applied to be effective at eliminating traces of the virus throughout the vehicle, especially at times when COVID-19 cases are peaking. Additionally, infectivity tests should be implemented to evaluate the efficiency of disinfection procedures to complement the information resulting from RT-PCR analysis. Modelling the probability of infection whilst travelling in buses under different scenarios indicates that forced ventilation greatly reduces the risk.