SARS-CoV-2 RNA Detection in Wastewater and Its Effective Correlation with Clinical Data during the Outbreak of COVID-19 in Salamanca.

Wastewater treatment plants (WWTPs) are the final stage of the anthropogenic water cycle where a wide range of chemical and biological markers of human activity can be found. In COVID-19 disease contexts, wastewater surveillance has been used to infer community trends based on viral abundance and SARS-CoV-2 RNA variant composition, which has served to anticipate and establish appropriate protocols to prevent potential viral outbreaks. Numerous studies worldwide have provided reliable and robust tools to detect and quantify SARS-CoV-2 RNA in wastewater, although due to the high dilution and degradation rate of the viral RNA in such samples, the detection limit of the pathogen has been a bottleneck for the proposed protocols so far. The current work provides a comprehensive and systematic study of the different parameters that may affect the detection of SARS-CoV-2 RNA in wastewater and hinder its quantification. The results obtained using synthetic viral RNA as a template allow us to consider that 10 genome copies per µL is the minimum RNA concentration that provides reliable and consistent values for the quantification of SARS-CoV-2 RNA. RT-qPCR analysis of wastewater samples collected at the WWTP in Salamanca (western Spain) and at six pumping stations in the city showed that below this threshold, positive results must be confirmed by sequencing to identify the specific viral sequence. This allowed us to find correlations between the SARS-CoV-2 RNA levels found in wastewater and the COVID-19 clinical data reported by health authorities. The close match between environmental and clinical data from the Salamanca case study has been confirmed by similar experimental approaches in four other cities in the same region. The present methodological approach reinforces the usefulness of wastewater-based epidemiology (WBE) studies in the face of future pandemic outbreaks.

Study of coliforms and Clostridium bacteria inactivation in wastewaters by a pilot photolysis process and by the maturation lagoons of a low-cost nature-based WWTP.

The inactivation processes of coliform bacteria (total and fecal) and sulphito-reducing Clostridium bacteria (vegetative species and spores) in water maturation lagoon of a low-cost nature-based wastewater treatment plant using constructed wetlands and through processes of photolysis in a pilot photoreactor have been comparatively studied. The different inactivation mechanisms by photolysis of these bacteria have been studied following the criteria of different statistical and kinetic models. Clostridium disinfection treatments fit models in which two types of bacteria populations coexist, one sensitive (vegetative species) and the other (spores) resistant to the treatment, the sensitive one (94%) with an inactivation rate of k = 0.24 ± 0.07 min-1 and the resistant one (6%) with k = 0.11 ± 0.05 min-1. Total coliform photolytic disinfection also shows two populations with different physiological state. The time required to reduce the first logarithmic decimal cycle of the different types of bacteria (physiological states) are δ1 = 4.2 ± 0.9 and δ2 = 8.3 ± 1.1 min, respectively. For fecal coliform photolytic disinfection, only bacteria population, with k = 1.15 ± 0.19 min-1, is found. The results obtained confirm the photolytic disinfection processes and maturation lagoon are effective systems for Clostridia bacteria removal after water treatment by nature-based systems. Total removal of coliform bacteria is not achieved by maturation lagoons, but their reduction is significant using low doses of cumulative radiation.