Genomic Analysis and Surveillance of Respiratory Syncytial Virus Using Wastewater-Based Epidemiology.

Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limit the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of severe acute respiratory syndrome coronavirus 2 in the community, but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying reverse-transcription quantitative polymerase chain reaction and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and nonpharmaceutical interventions.

Improved recovery of SARS-CoV-2 from wastewater through application of RNA and DNA stabilising agents.

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected.

Unprecedented Harmful algal bloom in the UK and Ireland's largest lake associated with gastrointestinal bacteria, microcystins and anabaenopeptins presenting an environmental and public health risk.

Harmful Algal Blooms (HABs) are outbreaks of aquatic toxic microalgae emerging as a global problem driven by nutrient enrichment, global climate change and invasive species. We uniquely describe a HAB of unprecedented duration, extent and magnitude during 2023 in Lough Neagh; the UK and Ireland's largest freshwater lake, using an unparalleled combination of satellite imagery, nutrient analysis, 16S rRNA gene sequencing and cyanotoxin profiling. The causative agent Microcystis aeruginosa accounted for over a third of DNA in water samples though common bacterioplankton species also bloomed. Water phosphate levels were hypertrophic and drove local algal biomass. The HAB pervaded the entire ecosystem with algal mats accumulating around jetties, marinas and lock gates. Over 80 % of bacterial DNA isolated from algal mat samples consisted of species associated with wildfowl or livestock faeces and human-effluent wastewater including 13 potential pathogens that can cause serious human illness including: E. coli, Salmonella, Enterobacter and Clostridium among others. Ten microcystins, nodularin and two anabaenopeptin toxins were confirmed as present (with a further microcystin and four anabaenopeptins suspected), with MC-RR and -LR in high concentrations at some locations (1,137-18,493 µg/L) with MC-LR exceeding World Health Organisation (WHO) recreational exposure guidelines in all algal mats sampled. This is the first detection of anabaenopeptins in any waterbody on the island of Ireland. Notwithstanding the ecological impacts, this HAB represented an environmental and public health risk, curtailing recreational activities in-and-around the lake and damaging local businesses. Reducing agricultural runoff and discharge from human-effluent wastewater treatment to manage nutrient loading, and the public health risk, should be the top priority of stakeholders, especially government. Key recommendations include Nature-based Solutions that avoid conflict with the productivity and profitability of the farming sector enhancing sustainability. We hope this stimulates real-world action to resolve the problems besetting this internationally important ecosystem.

Wastewater monitoring of human and avian influenza A viruses in Northern Ireland: a genomic surveillance study.

BACKGROUND: Influenza A viruses (IAVs) are significant pathogens of humans and other animals. Although endemic in humans and birds, novel IAV strains can emerge, jump species, and cause epidemics, like the latest variant of H5N1. Wastewater-based epidemiology (WBE) has been shown capable of detecting human IAVs. We aimed to assess whether whole-genome sequencing (WGS) of IAVs from wastewater is possible and can be used to discriminate between circulating strains of human and any non-human IAVs, such as those of avian origin. METHODS: Using a pan-IAV RT-quantitative PCR assay, six wastewater treatment works (WWTWs) across Northern Ireland were screened from Aug 1 to Dec 5, 2022. A nanopore WGS approach was used to sequence RT-qPCR-positive samples. Phylogenetic analysis of sequences relative to currently circulating human and non-human IAVs was performed. For comparative purposes, clinical data (PCR test results) were supplied by The Regional Virus Laboratory, Belfast Health and Social Care Trust (Belfast, Northern Ireland, UK). FINDINGS: We detected a dynamic IAV signal in wastewater from Sept 5, 2022, onwards across Northern Ireland, which did not show a clear positive relationship with the clinical data obtained for the region. Meta (mixed strain) whole-genome sequences were generated from wastewater samples displaying homology to only human and avian IAV strains. The relative proportion of IAV reads of human versus avian origin differed across time and sample site. A diversity in subtypes and lineages was detected (eg, H1N1, H3N2, and several avian). Avian segment 8 related to those found in recent H5N1 clade 2.3.4.4b was identified. INTERPRETATION: WBE affords a means to monitor circulating human and avian IAV strains and provide crucial genetic information. As such, WBE can provide rapid, cost-effective, year-round One Health surveillance to help control IAV epidemic and pandemic-related threats. However, optimisation of WBE protocols are necessary to ensure observed wastewater signals not only correlate with clinical case data, but yield information on the wider environmental pan-influenz-ome. FUNDING: Department of Health for Northern Ireland.

Detection of human adenovirus F41 in wastewater and its relationship to clinical cases of acute hepatitis of unknown aetiology.

As of 8 July 2022, the World Health Organization (WHO) have reported 1010 probable cases of acute hepatitis of unknown aetiology in children worldwide, including approximately 250 cases in the United Kingdom (UK). Clinical presentations have often been severe, with liver transplantation a frequent clinical outcome. Human adenovirus F41 (HAdV-F41) has been detected in most children with acute hepatitis, but its role in the pathogenesis of this infection has yet to be established. Wastewater-based epidemiology (WBE) has become a well-established tool for monitoring the community spread of SARS-CoV-2, as well as other pathogens and chemicals. In this study, we adopted a WBE approach to monitoring levels of HAdV-F40/41 in wastewater before and during an acute hepatitis outbreak in Northern Ireland. We report increasing detection of HAdV-F40/41 in wastewater, concomitant with increasing numbers of clinical cases. Amplicon whole genome sequencing further classified the wastewater-derived HAdV as belonging to the F41 genotype which in turn was homologous to clinically derived sequences. We propose that WBE has the potential to inform community surveillance of HAdV-F41 and can further contribute to the ongoing global discussion supporting HAdV-F41 involvement in acute hepatitis cases.