Reliability of wastewater analysis for monitoring COVID-19 incidence revealed by a long-term follow-up study

Background: Wastewater-based epidemiology has been used for monitoring human activities and waterborne pathogens. Although wastewaters can also be used for tracking SARS-CoV-2 at the population level, the reliability of this approach remains to be established, especially for early warning of outbreaks. Methods: We collected 377 samples from different treatment plants processing wastewaters of >1 million inhabitants in Valencia, Spain, between April 2020 and March 2021. Samples were cleaned, concentrated, and subjected to RT-qPCR to determine SARS-CoV-2 concentrations. These data were compared with cumulative disease notification rates over 7 and 14 day periods. Results: We amplified SARS-CoV-2 RNA in 75% of the RT-qPCRs, with an estimated detection limit of 100 viral genome copies per liter (gc/L). SARS-CoV-2 RNA concentration correlated strongly with disease notification rates over 14-day periods (Pearson r = 0.962, P < 0.001). A concentration >1000 gc/L showed >95% sensitivity and specificity as an indicator of more than 25 new cases per 100,000 inhabitants. Albeit with slightly higher uncertainty, these figures were reproduced using a 7-day period. Time series were similar for wastewaters data and declared cases, but wastewater RNA concentrations exhibited transient peaks that were not observed in declared cases and preceded major outbreaks by several weeks. Interpretation: Wastewater analysis provides a reliable tool for monitoring COVID-19, particularly at low incidence values, and is not biased by asymptomatic cases. Moreover, this approach might reveal previously unrecognized features of COVID-19 transmission.