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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 el

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.

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 elimi

SARS-CoV-2 was detected in Barcelona sewage long before the declaration of the first COVID-19 case, indicating that the infection was present in the population before the first imported case was reported. Sentinel surveillance of SARS-CoV-2 in wastewater would enable adoption of immediate measures in the event of future COVID-19 waves.