Systematic SARS-CoV-2 S-gene sequencing in wastewater samples enables early lineage detection and uncovers rare mutations in Portugal.

As the COVID-19 pandemic reached its peak, many countries implemented genomic surveillance systems to track the evolution and transmission of SARS-CoV-2. Transition from the pandemic to the endemic phase prioritized alternative testing strategies to maintain effective epidemic surveillance at the population level, with less intensive sequencing efforts. One such promising approach was Wastewater-Based Surveillance (WBS), which offers non-invasive, cost-effective means for analysing virus trends at the sewershed level. From 2020 onwards, wastewater has been recognized as an instrumental source of information for public health, with national and international authorities exploring options to implement national wastewater surveillance systems and increasingly relying on WBS as early warning of potential pathogen outbreaks. In Portugal, several pioneer projects joined the academia, water utilities and Public Administration around WBS. To validate WBS as an effective genomic surveillance strategy, it is crucial to collect long term performance data. In this work, we present one year of systematic SARS-CoV-2 wastewater surveillance in Portugal, representing 35 % of the mainland population. We employed two complementary methods for lineage determination - allelic discrimination by RT-PCR and S-gene sequencing. This combination allowed us to monitor variant evolution in near-real-time and identify low-frequency mutations. Over the course of this year-long study, spanning from May 2022 to April 2023, we successfully tracked the dominant Omicron sub-lineages, their progression and evolution, which aligned with concurrent clinical surveillance data. Our results underscore the effectiveness of WBS as a tracking system for virus variants, with the ability to unveil mutations undetected via massive sequencing of clinical samples from Portugal, demonstrating the ability of WBS to uncover new mutations and detect rare genetic variants. Our findings emphasize that knowledge of the genetic diversity of SARS-CoV-2 at the population level can be extended far beyond via the combination of routine clinical genomic surveillance with wastewater sequencing and genotyping.

Peracetic acid as a disinfectant for wastewater reuse - Regulation (EU) 2020/741 application on a pilot-scale.

Water scarcity affects already a large part of the world's population. To overcome this situation, water management is needed, and wastewater reuse must be implemented and included as a new approach. To achieve that objective water quality must comply with the parameters established in the Regulation (EU) 2020/741 of the European Parliament and the Council of the European Union and new treatment solutions have to be developed. The main goal of this pilot study was to evaluate the peracetic acid (PAA) disinfection efficiency in a real wastewater treatment plant (WWTP) in order to accomplish the wastewater reuse objective. To this end, six disinfection conditions were studied, three PAA doses (5, 10, and 15) and three contact times (5, 10, and 15) based on the commonly used disinfection operational conditions in real WWTP. Comparing the Total Suspended Solids (TSS), turbidity, Biological Oxygen Demand (BOD5) and Escherichia coli content, after and before the disinfection step, was possible to conclude that PAA ensures the Regulation (EU) 2020/741 requirements and that the disinfected effluent can be reused for several uses. All the conditions in which the PAA dose was 15 mg/L and the condition with 10 mg/L of PAA with a contact time of 15 min were the most promising, presenting the second highest water quality class achieved. The results of this study illustrate the potential of PAA as an alternative disinfectant for wastewater treatment and, bring it closer to the water reuse objective by presenting several possibilities for water uses.