Genomic Analysis and Surveillance of Respiratory Syncytial Virus (RSV) Using Wastewater-Based Epidemiology (WBE).

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 limits the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of SARS-CoV-2 in the community but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying RT-qPCR 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 non-pharmaceutical 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.

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.

Characterization of Bacteria Using Surface-Enhanced Raman Spectroscopy (SERS): Influence of Microbiological Factors on the SERS Spectra.

SERS is currently being explored as a rapid method for identification of bacteria but variation in the experimental procedures has resulted in considerable variation in the spectra reported for a range of bacterial species. Here, we show that mixing bacteria with a conventional citrate-reduced silver colloid (CRSC) and drying the resulting suspension yield highly reproducible spectra. These signals were due to intracellular components released when the structure of the bacteria was disrupted during sample preparation. This reproducibility allowed us to examine the effects of variables that do not arise in SERS of simple solutions but are relevant in studies of bacteria. These included growth phase and biological variation, which occurred when the same bacterial isolates were cultured under nominally identical conditions on different days. It was found that even under optimal standardized conditions the effect of differences in experimental parameters such as growth phase was very large in some bacterial species but insignificant in others. This suggests that it is important to avoid drawing general conclusions about bacterial SERS based on studies using small numbers of samples. Similarly, discrimination between bacterial species was straightforward when a small number of isolates with distinct spectral features were investigated; however, this became more challenging when more bacterial species were included, as this increased the possibility of finding different species of bacteria with similar spectra. These observations are important because they clearly delineate the challenges that will need to be addressed if SERS is to be used for clinical applications.