The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater produced interest in its use for sentinel surveillance at a community level and as a complementary approach to syndromic surveillance. With this work, we set the foundations for wastewater-based epidemiology (WBE) in Portugal by monitoring the trends of SARS-CoV-2 RNA circulation in the community, on a nationwide perspective during different epidemiological phases of the pandemic. The Charité assays (E_Sarbecco, RdRP, and N_Sarbecco) were applied to monitor, over 32-weeks (April to December 2020), the dynamics of SARS-CoV-2 RNA at the inlet of five wastewater treatment plants (WWTP), which together serve more than two million people in Portugal. Raw wastewater from three Coronavirus disease 2019 (COVID-19) reference hospitals was also analyzed during this period. In total, more than 600 samples were tested. For the first weeks, detection of SARS-CoV-2 RNA was sporadic, with concentrations varying from 10 3 to 10 5 genome copies per liter (GC/L). Prevalence of SARS-CoV-2 RNA increased steeply by the end of May into late June, mainly in Lisboa e Vale do Tejo region (LVT), during the reopening phase. After the summer, with the reopening of schools in mid-September and return to partial face-to-face work, a pronounced increase of SARS-CoV-2 RNA in wastewater was detected. In the LVT area, SARS-CoV-2 RNA load agreed with reported trends in hotspots of infection. Synchrony between trends of SARS-CoV-2 RNA in raw wastewater and daily new COVID-19 cases highlights the value of WBE as a surveillance tool, particularly after the phasing out of the epidemiological curve and when hotspots of disease re-emerge in the population which might be difficult to spot based solely on syndromic surveillance and contact tracing. This is the first study crossing several epidemiological stages highlighting the long-term use of WBE for SARS-CoV-2.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been extensively detected in raw wastewater in studies exploring wastewater-based epidemiology (WBE) for early warning purposes. Nonetheless, only a few limited studies investigated the presence of SARS-CoV-2 in treated wastewaters to determine the potential health risks across the water cycle. The detection of SARS-CoV-2 has been done mostly by RT-qPCR and ddPCR, which only provides information on the presence of nucleic acids rather than information on potential infectivity. In this study, we set to develop and evaluate the use of viability RT-qPCR for the selective discrimination and surveillance of infectious SARS-CoV-2 in secondary-treated wastewater. Enzymatic (nuclease) and viability dye (Reagent D) pretreatments were applied to infer infectivity through RT-qPCR using porcine epidemic diarrhea virus (PEDV) as a CoV surrogate. Infectivity tests were first performed on PEDV purified RNA, then on infectious and heat-inactivated PEDV, and finally on heat inactivated PEDV spiked in concentrated secondary-treated wastewater. The two viability RT-qPCR methods were then applied to 27 secondary-treated wastewater samples positive for SARS-CoV-2 RNA at the outlet of five large urban wastewater treatment plants in Portugal. Reagent D pretreatment showed similar behavior to cell culture for heat-inactivated PEDV and both viability RT-qPCR methods performed comparably to VERO E6 cell culture for SARS-CoV-2 present in secondary-treated wastewater, eliminating completely the RT-qPCR signal. Our study demonstrated the lack of infectious SARS-CoV-2 viral particles on secondary-treated wastewater through the application of two pretreatment methods for the rapid inference of infectivity through RT-qPCR, showing their potential application in environmental screening. This study addressed a knowledge gap on the public health risks of SARS-CoV-2 across the water cycle.
Over the last few decades, citizen awareness and perception of chemical products has been a topic of interest, particularly concerning national and international policy decision makers, expert/scientific platforms, and the European Union itself. To date, few qualitative studies on human biomonitoring have analysed communication materials, made recommendations in terms of biomonitoring surveillance, or asked for feedback in terms of specific biomonitoring methods. This paper provides in-depth insight on citizens’ perceptions of knowledge of biomonitoring, impact of chemical exposure on daily life, and claims on how results of research should be used. Four semi-structured focus groups were held in Austria, Portugal, Ireland, and the United Kingdom (UK). The cross-sectional observational qualitative design of this study allows for better understanding of public concern regarding chemicals, application, and use of human biomonitoring. The main findings of this study include citizens’ clear articulation on pathways of exposure, the demand on stakeholders for transparent decision-making, and sensitivity in communication of results to the public. Validated and trustful communication is perceived as key to empowering citizens to take action. The results can be used to facilitate decision-making and policy development, and feeds into the awareness needs of similar and future projects in human biomonitoring. Furthermore, it also brings to light ideas and concepts of citizens’ in shaping collaborative knowledge between citizens’, experts, scientists, and policy makers on equal terms.
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