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[Adapted from abstract]: This study investigated diurnal changes in SARS-CoV-2 and crAssphage concentrations, as well as physicochemical properties, in wastewater samples collected hourly from two wastewater treatment plants in Wales over 4 days. The aim was to establish if there were significant diurnal patterns that should be considered when using wastewater surveillance for SARS-CoV-2. The study also trialed three materials – tampons, filter paper, and cotton gauze – as passive wastewater samplers. The results showed SARS-CoV-2 and crAssphage were present in most samples, but concentrations remained largely stable over 24 hours. Some fluctuations in pH, orthophosphate, ammonium and turbidity levels were observed, typically increasing around midday when human activity is higher. Overall there were no significant diurnal changes in virus concentrations. The tampon passive samplers performed best, likely due to higher sorption capacity and durability. The authors conclude grab samples from wastewater influent tanks can provide representative alternatives to 24 hour composites for wastewater surveillance. While passive samplers are a viable low-cost approach, viral recovery needs optimisation depending on type. This study provides evidence to inform sampling strategies for SARS-CoV-2 wastewater surveillance programs.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]: Norovirus (NoV) is a highly contagious enteric virus that causes widespread outbreaks and a substantial number of deaths across communities. As clinical surveillance is often insufficient, wastewater-based epidemiology (WBE) may provide novel pathways of tracking outbreaks. To utilise WBE, it is important to use accurate and sensitive methods for viral quantification. In this study, we developed a one-step duplex RT-qPCR assay to simultaneously test the two main human pathogenic NoV genogroups, GI and GII, in wastewater samples. The assay had low limits of detection (LOD), namely 0.52 genome copies (gc)/µl for NoVGI and 1.37 gc/µl for NoVGII. No significant concentration-dependent interactions were noted for both NoVGI and for NoVGII when the two targets were mixed at different concentrations in the samples. When tested on wastewater-derived RNA eluents, no significant difference between duplex and singleplex concentrations were found for either target. Low levels of inhibition (up to 32 %) were noted due to organic matter present in the wastewater extracts. From these results we argue that the duplex RT-qPCR assay developed enables the sensitive detection of both NoVGI and NoVGII in wastewater-derived RNA eluents, in a time and cost-effective way and may be used for surveillance to monitor public and environmental health.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]: This study screened stool samples collected from infants attending selected hospitals in Nsukka for human enteric viruses and evaluated the seasonality of acute gastroenteritis (AGE) based on three-year records available at selected hospitals. The samples were analysed using an immunochromatographic lateral flow assay for diferential qualitative detection of rotavirus (RoV), adenovirus (AdV), and norovirus genogroups I and II (NoVI, NoVII). Three-year (2017–2019) retrospective data on the cases of AGE reported at the hospitals were also collected and analysed. The overall prevalence of acute gastroenteritis was high (75.83%), with 13.19% representing viral co-infections. Rotavirus detection rate (69.17%) was higher than that for other viral agents (15.83%). Both mono- and mixed infections were observed for RoV, AdV and NoVII, whereas NoVI was detected only in co-infection cases. Analysis of risk factors showed that acute gastroenteritis was detected more often in infants of age ˂1 year (73.53%) than in those 1≤2 years (22.55%) or>2 years (3.92%) in age. Gender and age were not associated with the cases of coinfections (p˂0.05). The seasonality data indicated one peak of the infection occurring in January 2017 which has decreased consecutively in the subsequent two years. These results demonstrate the prevalence and co-occurrence of enteric viruses in cases of infantile diarrhoea in Nsukka.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

 

[Adapted from abstract]: This study of UK adults quantified knowledge of COVID-19 symptoms, perceived health risk of contracting COVID-19, likelihood of returning to the UK with COVID-19 symptoms, likelihood to obey self-quarantining guidelines, how safe air travellers felt when flying during the pandemic, and perceptions towards face covering effectiveness. Overall knowledge of COVID-19 symptoms was poor. Men and younger age groups (18–44) were less informed than women and older age groups (44 +). A significant proportion (21%) of the population would likely travel back to the UK whilst displaying COVID-19 symptoms with many expressing that they would not fully comply with self-isolation guidelines. Overall, males and younger age groups had a reduced perceived personal risk from contracting COVID-19, posing a higher risk of transporting SARS-CoV-2 back to the UK. Poor passenger knowledge and behaviour undermines government guidelines and policies aimed at preventing SARS-CoV-2 entry into the UK. This supports the need for stricter, clearer and more targeted guidelines with point-of-departure viral testing and stricter quarantining upon arrival.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]: This manuscript showcases results from a large scale wastewater-based epidemiology (WBE) study focussed on multi-biomarker suite analysis of both chemical and biological determinants in 10 cities and towns across England equating to a population of ~7 million people. Population normalised daily loads (PNDLs) of many chemical markers were found, to a large extent, driven by the size of population contributing to wastewater (especially NCDs). An importance for tracking endogenous health markers as a generic marker of health status in communities was observed due to increased levels of HNE-MA seen at Barnoldswick wastewater treatment plant that coincided with higher-than-average paracetamol usage and SARS-CoV-2 prevalence in this community. PNDLs of virus markers were found to be highly variable. Being very prevalent in communities nationwide during sampling, SARS-CoV-2 presence in wastewater was to a large extent community driven. The same applies to the fecal marker virus, crAssphage, which is very prevalent in urban communities. In contrast, norovirus and enterovirus showed much higher variability in prevalence across all sites investigated, with clear cases of localised outbreaks in some cities while maintaining low prevalence in other locations. This study clearly demonstrates the potential for WBE to provide an integrated assessment of community health which can help target and validate policy interventions aimed at improving public health and wellbeing.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]: In this study, we monitored sewage in samples from terminals and aircraft at three major international airports in the UK for 1–3 weeks in March 2022. As the raw samples were more turbid than typical municipal wastewater, we used beef extract treatment followed by polyethylene glycol (PEG) precipitation to concentrate viruses, followed by reverse transcription quantitative PCR (RT-qPCR) for the detection of SARS-CoV-2 and a faecal indicator virus, crAssphage. All samples taken from sewers at the arrival terminals of Heathrow and Bristol airports, and 85% of samples taken from sites at Edinburgh airport, were positive for SARS-CoV-2. This suggests a high COVID-19 prevalence among passengers and/or airport staff members. Samples derived from aircraft also showed 93% SARS-CoV-2 positivity. No difference in viral prevalence was found before and after COVID-19 travel restrictions were lifted. Our results suggest that WBE is a useful tool for monitoring the global transfer rate of human pathogens and other disease-causing agents across international borders and should form part of wider international efforts to monitor and contain the spread of future disease outbreaks.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]:  In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]: Here, we evaluated the laboratory and field performance of 8 passive sampler materials (NanoCeram, ZetaPlus, nylon and ion exchange membranes, cellulose acetate filters, glass wool, cotton-based Moore swabs and tampons) to capture viruses and bacteria from wastewater. Viral capture focused on SARS-CoV-2, the bacteriophage Phi6 and the faecal marker virus, crAssphage. We showed that the best performing passive sampler in terms of cost, ease of deployment and viral capture were the electronegative cotton-based swabs and tampons. We speculate that viral capture is a combination of trapping of particulate matter to which viruses are attached, as well as electrostatic attraction of viral particles from solution. The patterns of viral capture across the different sampling materials were similar providing evidence that they can be used to monitor multiple public health targets. The types of bacteria trapped by the passive samplers were material-specific, but possessed a different 16S rRNA gene profile to the wastewater, suggesting preferential retention of specific bacteria. We conclude that the choice of passive sampler and deployment time greatly influences the pattern and amount of viral and bacterial capture.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]:  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. Furthermore, wastewater and clinical sequencing match well in the estimated timing of new variant rises and the first detection of a new variant in a given area may occur in either clinical or wastewater samples. The correspondence between wastewater and clinical variant frequencies demonstrates the reliability of wastewater surveillance. However, discrepancies in the first detection of the Alpha variant between the two approaches highlight that wastewater monitoring can also capture missing information, possibly resulting from asymptomatic cases or communities less engaged with testing programmes, as found by a simultaneous surge testing effort across the city.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract]: In this study of UK adults (n = 2103), we quantified the likelihood of defecation prior to departure, on the aircraft and upon arrival on both short- and long-haul flights. The results were then used to assess the likelihood of capturing the signal from infected individuals at UK travel hubs. We found that an individual’s likelihood to defecate on short-haul flights was low, but was higher on long-haul flights. This behaviour pattern was higher among males and younger age groups. The maximum likelihood of defecation was prior to departure. Based on known SARS-CoV-2 faecal shedding rates (30–60 %) and an equal probability of infected individuals being on short- (71 % of inbound flights) and long-haul flights (29 %), we estimate that aircraft wastewater is likely to capture ca. 8–14 % of SARS-CoV-2 cases entering the UK. Monte Carlo simulations predicted that SARS-CoV-2 would be present in wastewater on 14 % of short-haul flights and 62 % of long-haul flights under current pandemic conditions. We conclude that aircraft wastewater alone is insufficient to effectively monitor all the transboundary entries of faecal-borne pathogens but can form part of a wider strategy for public heath surveillance at national borders.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[From abstract]: We reflect on the current knowledge of WBE for these applications and identify barriers and opportunities for expanding beyond SARS-CoV-2. This paper critically reviews the applications of WBE for public health and identifies the important research gaps for WBE to be a useful tool in public health. It considers possible uses for pathogenic viruses, AMR, and chemicals. It summarises the current evidence on the following: (1) the presence of markers in stool and urine; (2) environmental factors influencing persistence of markers in wastewater; (3) methods for sample collection and storage; (4) prospective methods for detection and quantification; (5) reducing uncertainties; and (6) further considerations for public health use.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[From abstract]:  In this study, we compared the performance of five commonly used wastewater concentration methods for the detection and quantification of pathogenic viruses (SARS-CoV-2, norovirus, rotavirus, influenza, and measles viruses), fecal indicator viruses (crAssphage, adenovirus, pepper mild mottle virus), and process control viruses (murine norovirus and bacteriophage Phi6) in laboratory spiking experiments. The methods evaluated included those based on either ultrafiltration (Amicon centrifugation units and InnovaPrep device) or precipitation (using polyethylene glycol [PEG], beef extract-enhanced PEG, and ammonium sulfate). The two best methods were further tested on 115 unspiked wastewater samples. We found that the volume and composition of the wastewater and the characteristics of the target viruses greatly affected virus recovery, regardless of the method used for concentration. All tested methods are suitable for routine virus concentration; however, the Amicon ultrafiltration method and the beef extract-enhanced PEG precipitation methods yielded the best recoveries.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[From abstract]: In this study, we compared the virus-binding capacity of two passive samplers (cotton-based tampons and ion exchange filter papers) in two different water types (deionised water and wastewater). Here we focused on the capture of wastewater-associated viruses including Influenza A and B (Flu-A & B), SARS-CoV-2, human adenovirus (AdV), norovirus GII (NoVGII), measles virus (MeV), pepper mild mottle virus (PMMoV), the faecal marker crAssphage and the process control virus Pseudomonas virus phi6. After deployment, we evaluated four different methods to recover viruses from the passive samplers namely, (i) phosphate buffered saline (PBS) elution followed by polyethylene glycol (PEG) precipitation, (ii) beef extract (BE) elution followed by PEG precipitation, (iii) no-elution into PEG precipitation, and (iv) direct extraction. We found that the tampon-based passive samplers had higher viral recoveries in comparison to the filter paper. Overall, the preferred viral recovery method from the tampon passive samplers was the no-elution/PEG precipitation method.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract] Using urban wastewater samples, we tested three viral concentration methods; polyethylene glycol precipitation (PEG), ammonium sulphate precipitation (AS), and CP select™ InnovaPrep® (IP) ultrafiltration. Our results show that choice of methodology had small effect on viral recovery of SARS-CoV-2 and crAssphage in wastewater samples within this study. In contrast, sample turbidity, storage temperature, and surfactant load did affect viral recovery, highlighting the need for careful consideration of the viral concentration methodology used when working with wastewater samples.

 

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[From abstract] In this paper a discussion of measurement uncertainty associated with surveillance of wastewater, focusing on lessons-learned from the UK programmes monitoring COVID-19 is presented, showing that sources of uncertainty impacting measurement quality and interpretation of data for public health decision-making, are varied and complex. While some factors remain poorly understood, we present approaches taken by the UK programmes to manage and mitigate the more tractable sources of uncertainty. This work provides a platform to integrate uncertainty management into WBE activities as part of global One Health initiatives beyond the pandemic.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[From Abstract] We report here the development of a national WBE program across 28 WWTPs serving 50% of the population of Scotland, including large conurbations, as well as low-density rural and remote island communities. For each WWTP catchment area, we quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases. Daily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation (ρ > 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 25 cases for larger plants to a single case in smaller plants.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

In this work, we used a recently discovered human gut bactreiophage, the crAssphage, to evaluate wastewater contamination in river and estuarine water, sediment and mussel. The crAssphage was found in untreated and treates wastewater at extremely high concentrations and detected in the contaminated water, sediment and mussel. Data suggest that the crAssphage may be a valuable tool to detect wastewater contamination in the environment.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

[Adapted from abstract] In this paper we evaluated diurnal pattern in enteric virus (norovirus, sapovirus and adenovirus) concentrations in treated and untreated wastewater collected bihourly for three days, in three seasons at three locations.  Adenovirus titre was high and relatively constant in all samples, whereas the NoVGI, NoVGII and SaVGI showed high concentrations during autumn and winter and low counts during the summer. Diurnal patterns were detected in pH and turbidity for some sampling periods. Our findings suggest that viral particle number in wastewater is not affected by daily chemical fluctuations. Hence, a grab sample taken at any point during the day may be sufficient to enumerate the viral load of wastewater effluent within an order of magnitude while four samples a day are recommended for testing wastewater influent samples.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

Here we describe a detailed protocol for the concentration of river, estuarine water and treated and raw wastewater concentration for the detection and quantification of viruses. The method comprises of a primary concentration using tangential flow ultrafiltration followed by elution and secondary precipitation to reduce the volume to 2 mL. We have carefully validated this method and have used for the surveillance of enteric viruses in wastewaters, in surface water samples contaminated with wastewater (> 150 samples). The method is also suitable for the concentration of large volumes (up to 100 L) of surface water for metagenomics approaches.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

 

[From abstract] In this study, we investigated the use of viromics, i.e., high-throughput sequencing of the biosphere’s viral fraction, to detect human-/animal-pathogenic RNA viruses in the Conwy river catchment area in Wales, United Kingdom. We found a higher richness of RNA viruses in wastewater samples than in river water and sediment, and we assembled a complete norovirus genotype GI.2 genome from wastewater effluent, which was not contemporaneously detected by conventional reverse transcription-quantitative PCR (qRT-PCR). The simultaneous presence of diverse rotavirus signatures in wastewater indicated the potential for zoonotic infections in the area and suggested runoff from pig farms as a possible origin of these viruses. Our results show that viromics can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided appropriate and rigorous controls are included.

Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

This paper analyses the distribution of enteric viruses in the Conwy River and estuary. We have validated a novel two-step concentration method suitable for low and high turbidity (e.g. wastewater), freshwater and seawater samples. We used this and previously established methods for the 1-year surveillance of enteric viruses in wastewater, the receiving river and estuarine water, sediment and shellfish samples. For the first time, we found sapovirus (enteric virus causing gastroenteritis with symptoms similar to norovirus infection) in UK wastewater samples. We also found norovirus in high concentrations during local gastroenteritis outbreaks in all sample types. We also found that due to their high concentrations and lack of seasonal distribution, adenoviruses can be suitable indicators for wastewater contamination tracking in the water environment. Full references to the paper can be seen on our publications page http://www.viraqua.uk/publications/.

The second paper to be publish from the VIRAQUA project has been published and is now available to download (http://link.springer.com/article/10.1007/s12560-017-9293-5).  In this study, the authors developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis, along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency.  It was concluded that ‘these assays allow the thorough assessment of the target viruses in sediment and shellfish samples and are useful for quantitative risk assessment of wastewater-contaminated environments’.   A full reference for this paper can be found on our publications page.

 

 

 

A collaborative paper, co-authored by Davey Jones, Shelagh Malham and others from Bangor University, Liverpool University, Natural Resources Wales and Bangor Mussel Producers Ltd. reviewing the risk of norovirus infection from the consumption of shellfish had been published in Food and Environmental Virology.  Conclusions are drawn as to the likely impact of climate change and shellfish role in the transmission of norovirus to the human population.  A full reference for this paper can be found on our publications page.

 

 

 

 

The new year kicked off with the first Viraqua project publication with Kata Farkas, Francis Hassard, James McDonald, Shelagh Malham and Davey Jones all co-authors on a paper in Frontiers in Microbiology evaluating molecular methods for the detection and quantification of pathogen-derived nucleic acids in sediment.” Full references can be found on our publications
page.

 

 

 

The last week has seen two new project publications; David Lees is co-author of a paper modelling risk factors for norovirus contamination of shellfish water http://dx.doi.org/10.1016/j.ijfoodmicro.2016.10.028 whilst Kata Farkas, Davey Jones, Shelagh Malham and James McDonald are all co-authors of a review paper addressing the abundance and distribution of enteric bacteria and viruses in coastal and estuarine sediments http://dx.doi.org/10.3389/fmicb.2016.01692  Full references to both papers can be seen on our publications page http://www.viraqua.uk/publications/.