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

Diurnal changes in pathogenic and indicator virus concentrations in wastewater

Wastewater-based epidemiology (WBE) has been commonly used for monitoring SARS-CoV-2 outbreaks. As sampling times and methods (i.e. grab vs composite) may vary, diurnal changes of viral concentrations in sewage should be better understood. In this study, we collected untreated wastewater samples hourly for 4 days at two wastewater treatment plants in Wales to establish diurnal patterns in virus concentrations and the physico-chemical properties of the water. Simultaneously, we also trialled three absorbent materials as passive samples as a simple and cost-efficient alternative for the collection of composite samples. Ninety-six percent of all liquid samples (n = 74) and 88% of the passive samplers (n = 59) were positive for SARS-CoV-2, whereas 87% and 97% of the liquid and passive samples were positive for the faecal indicator virus crAssphage, respectively. We found no significant daily variations in the concentration of the target viruses, ammonium and orthophosphate, and the pH and electrical conductivity levels were also stable. Weak positive correlations were found between some physico-chemical properties and viral concentrations. More variation was observed in samples taken from the influent stream as opposed to those taken from the influent tank. Of the absorbent materials trialled as passive samples, we found that tampons provided higher viral recoveries than electronegative filter paper and cotton gauze swabs. For all materials tested, viral recovery was dependent on the virus type. Our results indicate that grab samples may provide representative alternatives to 24-h composite samples if taken from the influent tank, hence reducing the costs of sampling for WBE programmes. Tampons are also viable alternatives for cost-efficient sampling; however, viral recovery should be optimised prior to use

Diurnal changes in pathogenic and indicator virus concentrations in wastewater

Wastewater-based epidemiology (WBE) has been commonly used for monitoring SARS-CoV-2 outbreaks. As sampling times and methods (i.e. grab vs composite) may vary, diurnal changes of viral concentrations in sewage should be better understood. In this study, we collected untreated wastewater samples hourly for 4 days at two wastewater treatment plants in Wales to establish diurnal patterns in virus concentrations and the physico-chemical properties of the water. Simultaneously, we also trialled three absorbent materials as passive samples as a simple and cost-efficient alternative for the collection of composite samples. Ninety-six percent of all liquid samples (n = 74) and 88% of the passive samplers (n = 59) were positive for SARS-CoV-2, whereas 87% and 97% of the liquid and passive samples were positive for the faecal indicator virus crAssphage, respectively. We found no significant daily variations in the concentration of the target viruses, ammonium and orthophosphate, and the pH and electrical conductivity levels were also stable. Weak positive correlations were found between some physico-chemical properties and viral concentrations. More variation was observed in samples taken from the influent stream as opposed to those taken from the influent tank. Of the absorbent materials trialled as passive samples, we found that tampons provided higher viral recoveries than electronegative filter paper and cotton gauze swabs. For all materials tested, viral recovery was dependent on the virus type. Our results indicate that grab samples may provide representative alternatives to 24-h composite samples if taken from the influent tank, hence reducing the costs of sampling for WBE programmes. Tampons are also viable alternatives for cost-efficient sampling; however, viral recovery should be optimised prior to use

Examining the stability of viral RNA and DNA in wastewater: Effects of storage time, temperature, and freeze-thaw cycles

Wastewater-based epidemiology (WBE) has been demonstrably successful as a relatively unbiased tool for monitoring levels of SARS-CoV-2 virus circulating in communities during the COVID-19 pandemic. Accumulated biobanks of wastewater samples allow retrospective exploration of spatial and temporal trends for public health indicators such as chemicals, viruses, antimicrobial resistance genes, and the possible emergence of novel human or zoonotic pathogens. We investigated virus resilience to time, temperature, and freeze-thaw cycles, plus the optimal storage conditions to maintain the stability of genetic material (RNA/DNA) of viral +ssRNA (Envelope – E, Nucleocapsid – N and Spike protein – S genes of SARS-CoV-2), dsRNA (Phi6 phage) and circular dsDNA (crAssphage) in wastewater. Samples consisted of (i) processed and extracted wastewater samples, (ii) processed and extracted distilled water samples, and (iii) raw, unprocessed wastewater samples. Samples were stored at –80 °C, –20 °C, 4 °C, or 20 °C for 10 days, going through up to 10 freeze-thaw cycles (once per day). Sample stability was measured using reverse transcription quantitative PCR, quantitative PCR, automated electrophoresis, and short-read whole genome sequencing. Exploring different areas of the SARS-CoV-2 genome demonstrated that the S gene in processed and extracted samples showed greater sensitivity to freeze-thaw cycles than the E or N genes. Investigating surrogate and normalisation viruses showed that Phi6 remains a stable comparison for SARS-CoV-2 in a laboratory setting and crAssphage was relatively resilient to temperature variation. Recovery of SARS-CoV-2 in raw unprocessed samples was significantly greater when stored at 4 °C, which was supported by the sequencing data for all viruses – both time and freeze-thaw cycles negatively impacted sequencing metrics. Historical extracts stored at –80 °C that were re-quantified 12, 14 and 16 months after original quantification showed no major changes. This study highlights the importance of the fast processing and extraction of wastewater samples, following which viruses are relatively robust to storage at a range of temperatures

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

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%–21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing. [Abstract copyright: © The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.