Poor air passenger knowledge of COVID-19 symptoms and behaviour undermines strategies aimed at preventing the import of SARS-CoV-2 into the UK.

Air travel mediates transboundary movement of SARS-CoV-2. To prepare for future pandemics, we sought to understand air passenger behaviour and perceived risk during the COVID-19 pandemic. This study of UK adults (n = 2103) 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 (n = 305), 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.

Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR.

Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. 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. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.

Predicting the dispersal of SARS-CoV-2 RNA from the wastewater treatment plant to the coast.

Viral pathogens including SARS-CoV-2 RNA have been detected in wastewater treatment effluent, and untreated sewage overflows, that pose an exposure hazard to humans. We assessed whether SARS-CoV-2 RNA was likely to have been present in detectable quantities in UK rivers and estuaries during the first wave of the Covid-19 pandemic. We simulated realistic viral concentrations parameterised on the Camel and Conwy catchments (UK) and their populations, showing detectable SARS-CoV-2 RNA concentrations for untreated but not for treated loading, but also being contingent on viral decay, hydrology, catchment type/shape, and location. Under mean or low river flow conditions, viral RNA concentrated within the estuaries allowing for viral build-up and caused a lag by up to several weeks between the peak in community infections and the viral peak in the environment. There was an increased hazard posed by SARS-CoV-2 RNA with a T 90 decay rate >24 h, as the estuarine build-up effect increased. High discharge events transported the viral RNA downstream and offshore, increasing the exposure risk to coastal bathing waters and shellfisheries - although dilution in this case reduced viral concentrations well below detectable levels. Our results highlight the sensitivity of exposure to viral pathogens downstream of wastewater treatment, across a range of viral loadings and catchment characteristics - with implications to environmental surveillance.

Suitability of aircraft wastewater for pathogen detection and public health surveillance.

International air travel is now widely recognised as one of the primary mechanisms responsible for the transnational movement and global spread of SARS-CoV-2. Monitoring the viral load and novel lineages within human-derived wastewater collected from aircraft and at air transport hubs has been proposed as an effective way to monitor the importation frequency of viral pathogens. The success of this approach, however, is highly dependent on the bathroom and defecation habits of air passengers during their journey. 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. To obtain a representative cross-section of the population, the survey was stratified by geographical region, gender, age, parenting status, and social class. We found that an individual's likelihood to defecate on short-haul flights (< 6 h in duration) was low (< 13 % of the total), but was higher on long-haul flights (< 36 %; > 6 h in duration). This behaviour pattern was higher among males and younger age groups. The maximum likelihood of defecation was prior to departure (< 39 %). 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.

Critical Evaluation of Different Passive Sampler Materials and Approaches for the Recovery of SARS-CoV-2, Faecal-Indicator Viruses and Bacteria from Wastewater

During the COVID-19 pandemic, wastewater-based epidemiology (WBE) has proven to be an effective tool for monitoring the prevalence of SARS-CoV-2 in urban communities. However, low-cost, simple, and reliable wastewater sampling techniques are still needed to promote the widespread adoption of WBE in many countries. Since their first use for public health surveillance in the 1950s, many types of passive samplers have been proposed, however, there have been few systematic studies comparing their ability to co-capture enveloped viruses and bacteria. 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. When deployed at wastewater treatment plants, the passive samplers worked best up to 6 h, after which they became saturated or exhibited a loss of virus, probably due to night-time wash-out. 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.

Suitability of aircraft wastewater for pathogen detection and public health surveillance

International air travel is now widely recognised as one of the primary mechanisms responsible for the transnational movement and global spread of SARS-CoV-2. Monitoring the viral load and novel lineages within human-derived wastewater collected from aircraft and at air transport hubs has been proposed as an effective way to monitor the importation frequency of viral pathogens. The success of this approach, however, is highly dependent on the bathroom and defecation habits of air passengers during their journey. 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. To obtain a representative cross-section of the population, the survey was stratified by geographical region, gender, age, parenting status, and social class. We found that an individual's likelihood to defecate on short-haul flights (< 6 h in duration) was low (< 13 % of the total), but was higher on long-haul flights (< 36 %;> 6 h in duration). This behaviour pattern was higher among males and younger age groups. The maximum likelihood of defecation was prior to departure (< 39 %). 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. Graphical Unlabelled Image

Comparative Assessment of Filtration- and Precipitation-Based Methods for the Concentration of SARS-CoV-2 and Other Viruses from Wastewater.

Wastewater-based epidemiology (WBE) has been widely used to track levels of SARS-CoV-2 infection in the community during the COVID-19 pandemic. Due to the rapid expansion of WBE, many methods have been used and developed for virus concentration and detection in wastewater. However, very little information is available on the relative performance of these approaches. 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. We recommend the use of ultrafiltration-based concentration for low sample volumes with high virus titers and ammonium levels and the use of precipitation-based concentration for rare pathogen detection in high-volume samples. IMPORTANCE As wastewater-based epidemiology is utilized for the surveillance of COVID-19 at the community level in many countries, it is crucial to develop and validate reliable methods for virus detection in sewage. The most important step in viral detection is the efficient concentration of the virus particles and/or their genome for subsequent analysis. In this study, we compared five different methods for the detection and quantification of different viruses in wastewater. We found that dead-end ultrafiltration and beef extract-enhanced polyethylene glycol precipitation were the most reliable approaches. We also discovered that sample volume and physico-chemical properties have a great effect on virus recovery. Hence, wastewater process methods and start volumes should be carefully selected in ongoing and future wastewater-based national surveillance programs for COVID-19 and beyond.

Assessment of two types of passive sampler for the efficient recovery of SARS-CoV-2 and other viruses from wastewater.

Wastewater-based epidemiology (WBE) has proven to be a useful surveillance tool during the ongoing SARS-CoV-2 pandemic, and has driven research into evaluating the most reliable and cost-effective techniques for obtaining a representative sample of wastewater. When liquid samples cannot be taken efficiently, passive sampling approaches have been used, however, insufficient data exists on their usefulness for multi-virus capture and recovery. 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. Furthermore, we evidenced that non-enveloped viruses had higher percent recoveries from the passive samplers than enveloped viruses. This is the first study of its kind to assess passive sampler and viral recovery methods amongst a plethora of viruses commonly found in wastewater or used as a viral surrogate in wastewater studies.

A comparison of precipitation and filtration-based SARS-CoV-2 recovery methods and the influence of temperature, turbidity, and surfactant load in urban wastewater.

Wastewater-based epidemiology (WBE) has become a complimentary surveillance tool during the SARS-CoV-2 pandemic. Viral concentration methods from wastewater are still being optimised and compared, whilst viral recovery under different wastewater characteristics and storage temperatures remains poorly understood. Using urban wastewater samples, we tested three viral concentration methods; polyethylene glycol precipitation (PEG), ammonium sulphate precipitation (AS), and CP select™ InnovaPrep® (IP) ultrafiltration. We found no major difference in SARS-CoV-2 and faecal indicator virus (crAssphage) recovery from wastewater samples (n = 46) using these methods, PEG slightly (albeit non-significantly), outperformed AS and IP for SARS-CoV-2 detection, as a higher genome copies per litre (gc/l) was recorded for a larger proportion of samples. Next generation sequencing of 8 paired samples revealed non-significant differences in the quality of data between AS and IP, though IP data quality was slightly better and less variable. A controlled experiment assessed the impact of wastewater suspended solids (turbidity; 0-400 NTU), surfactant load (0-200 mg/l), and storage temperature (5-20 °C) on viral recovery using the AS and IP methods. SARS-CoV-2 recoveries were >20% with AS and <10% with IP in turbid samples, whilst viral recoveries for samples with additional surfactant were between 0-18% for AS and 0-5% for IP. Turbidity and sample storage temperature combined had no significant effect on SARS-CoV-2 recovery (p > 0.05), whilst surfactant and storage temperature combined were significant negative correlates (p < 0.001 and p < 0.05, respectively). In conclusion, 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.