City-wide wastewater genomic surveillance through the successive emergence of SARS-CoV-2 Alpha and Delta variants

Genomic surveillance of SARS-CoV-2 has been essential to provide an evidence base for public health decisions throughout the SARS-CoV-2 pandemic. Sequencing data from clinical cases has provided data crucial to understanding disease transmission and the detection, surveillance, and containment of outbreaks of novel variants, which continue to pose fresh challenges. However, genomic wastewater surveillance can provide important complementary information by providing estimates of variant frequencies which do not suffer from sampling bias, and capturing all variants circulating in a population. Here we show that genomic SARS-CoV-2 wastewater surveillance can detect fine-scale differences within urban centres, specifically within the city of Liverpool, UK, during the emergence of Alpha and Delta variants between November 2020 and June 2021. Overall, the correspondence between wastewater and clinical variant frequencies demonstrates the reliability of wastewater surveillance. Yet, discrepancies between the two approaches when the Alpha variant was first detected emphasises that wastewater monitoring can also capture missing information resulting from asymptomatic cases or communities less engaged with testing programmes, as found by a simultaneous surge testing effort across the city.

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

The COVID-19 pandemic continues to pose a threat to the general population. The ongoing vaccination programs provide protection to individuals and facilitate the opening of society and a return to normality. However, emergent and existing SARS-CoV-2 variants capable of evading the immune system endanger the efficacy of the vaccination strategy. To preserve the efficacy of SARS-CoV-2 vaccination globally, aggressive and effective surveillance for known and emerging SARS-CoV-2 Variants of Concern (VOC) is required. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. In this work, we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to allow rapid and sensitive discrimination of VOCs, that can be used at point of care and/or implemented in the pipelines of small or large testing facilities, and even determine proportion of VOCs in pooled population-level wastewater samples. This technology aims to complement the ongoing sequencing efforts to allow facile and, crucially, rapid identification of individuals infected with VOCs to help break infection chains. Here, we show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants. In addition, we show the rapid adaptability of the technique for new and emerging VOCs such as Omicron. Short abstractThe COVID-19 pandemic continues to pose a threat as emergent and existing SARS-CoV-2 variants endanger the efficacy of the vaccination strategy. Rapid surveillance for known and emerging SARS-CoV-2 Variants of Concern (VOC) would be assisted by effective molecular diagnostics procedures. Here we develop the recent SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) for rapid and sensitive discrimination of VOCs to complement sequencing and allow rapid identification of individuals infected with VOC. We show our assays can be implemented with test samples in the pipelines of large testing facilities, as well as determine the proportion of VOCs in pooled population level wastewater samples and has potential applicability at point of care. We demonstrate the optimisation of new VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validate these with samples from the Cardiff University Testing Service, as well as the applicability of VarLOCK to national-level wastewater surveillance of SARS-CoV-2 variants. We also demonstrate the rapid adaptability of the technique for new and emerging VOCs such as Omicron.

COVID-19 mass testing: harnessing the power of wastewater epidemiology

BackgroundCOVID-19 patients shed SARS-CoV-2 RNA in their faeces. We hypothesised that detection of SARS-CoV-2 RNA in wastewater treatment plant (WWTP) influent could be a valuable tool to assist in public health decision making. We aimed to rapidly develop and validate a scalable methodology for the detection of SARS-CoV-2 RNA in wastewater that could be implemented at a national level and to determine the relationship between the wastewater signal and COVID-19 cases in the community. MethodsWe developed a filtration-based methodology for the concentration of SARS-CoV-2 from WWTP influent and subsequent detection and quantification by RT-qPCR. This methodology was used to monitor 28 WWTPs across Scotland, serving 50% of the population. For each WWTP catchment area, we collected data describing COVID-19 cases and deaths. We quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases. FindingsDaily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation ({rho}>0.9) with COVID-19 cases within a catchment. As the incidence of COVID-19 cases within a community increased, a linear relationship emerged between cases and influent viral RNA load. There were significant differences between WWTPs in their capacity to predict case numbers based on influent viral RNA load, with the limit of detection ranging from twenty-five cases for larger plants to a single case in smaller plants. InterpretationThe levels of SARS-CoV-2 RNA in WWTP influent provide a cost-effective and unbiased measure of COVID-19 incidence within a community, indicating that national scale wastewater-based epidemiology can play a role in COVID-19 surveillance. In Scotland, wastewater testing has been expanded to cover 75% of the population, with sub-catchment sampling being used to focus surge testing. SARS-CoV-2 variant detection, assessment of vaccination on community transmission and surveillance for other infectious diseases represent promising future applications. FundingThis study was funded by project grants from the Scottish Government via the Centre of Expertise for Waters (CD2019/06) and The Natural Environment Research Councils COVID-19 Rapid Response grants (NE/V010441/1). The Roslin Institute receives strategic funding from the Biotechnology and Biological Sciences Research Council (BB/P013740/1, BBS/E/D/20002173). Sample collection and supplementary analysis was funded and undertaken by Scottish Water and the majority of the sample analysis was funded and undertaken by the Scottish Environment Protection Agency.