Direct-ink-write cross-linkable bottlebrush block copolymers for on-the-fly control of structural color.

Additive manufacturing capable of controlling and dynamically modulating structures down to the nanoscopic scale remains challenging. By marrying additive manufacturing with self-assembly, we develop a UV (ultra-violet)-assisted direct ink write approach for on-the-fly modulation of structural color by programming the assembly kinetics through photo-cross-linking. We design a photo-cross-linkable bottlebrush block copolymer solution as a printing ink that exhibits vibrant structural color (i.e., photonic properties) due to the nanoscopic lamellar structures formed post extrusion. By dynamically modulating UV-light irradiance during printing, we can program the color of the printed material to access a broad spectrum of visible light with a single ink while also creating color gradients not previously possible. We unveil the mechanism of this approach using a combination of coarse-grained simulations, rheological measurements, and structural characterizations. Central to the assembly mechanism is the matching of the cross-linking timescale with the assembly timescale, which leads to kinetic trapping of the assembly process that evolves structural color from blue to red driven by solvent evaporation. This strategy of integrating cross-linking chemistry and out-of-equilibrium processing opens an avenue for spatiotemporal control of self-assembled nanostructures during additive manufacturing.

Investigation of the yielding transition in concentrated colloidal systems via rheo-XPCS.

We probe the microstructural yielding dynamics of a concentrated colloidal system by performing creep/recovery tests with simultaneous collection of coherent scattering data via X-ray Photon Correlation Spectroscopy (XPCS). This combination of rheology and scattering allows for time-resolved observations of the microstructural dynamics as yielding occurs, which can be linked back to the applied rheological deformation to form structure-property relations. Under sufficiently small applied creep stresses, examination of the correlation in the flow direction reveals that the scattering response recorrelates with its predeformed state, indicating nearly complete microstructural recovery, and the dynamics of the system under these conditions slows considerably. Conversely, larger creep stresses increase the speed of the dynamics under both applied creep and recovery. The data show a strong connection between the microstructural dynamics and the acquisition of unrecoverable strain. By comparing this relationship to that predicted from homogeneous, affine shearing, we find that the yielding transition in concentrated colloidal systems is highly heterogeneous on the microstructural level.

Near-source passive sampling for monitoring viral outbreaks within a university residential setting.

Wastewater-based epidemiology (WBE) has proven to be a powerful tool for the population-level monitoring of pathogens, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For assessment, several wastewater sampling regimes and methods of viral concentration have been investigated, mainly targeting SARS-CoV-2. However, the use of passive samplers in near-source environments for a range of viruses in wastewater is still under-investigated. To address this, near-source passive samples were taken at four locations targeting student hall of residence. These were chosen as an exemplar due to their high population density and perceived risk of disease transmission. Viruses investigated were SARS-CoV-2 and its variants of concern (VOCs), influenza viruses, and enteroviruses. Sampling was conducted either in the morning, where passive samplers were in place overnight (17 h) and during the day, with exposure of 7 h. We demonstrated the usefulness of near-source passive sampling for the detection of VOCs using quantitative polymerase chain reaction (qPCR) and next-generation sequencing (NGS). Furthermore, several outbreaks of influenza A and sporadic outbreaks of enteroviruses (some associated with enterovirus D68 and coxsackieviruses) were identified among the resident student population, providing evidence of the usefulness of near-source, in-sewer sampling for monitoring the health of high population density communities.

Sustained detection of type 2 poliovirus in London sewage between February and July, 2022, by enhanced environmental surveillance.

BACKGROUND: The international spread of poliovirus exposes all countries to the risk of outbreaks and is designated a Public Health Emergency of International Concern by WHO. This risk can be exacerbated in countries using inactivated polio vaccine, which offers excellent protection against paralysis but is less effective than oral vaccine against poliovirus shedding, potentially allowing circulation without detection of paralytic cases for long periods of time. Our study investigated the molecular properties of type 2 poliovirus isolates found in sewage with an aim to detect virus transmission in the community. METHODS: We performed environmental surveillance in London, UK, testing sewage samples using WHO recommended methods that include concentration, virus isolation in cell culture, and molecular characterisation. We additionally implemented direct molecular detection and determined whole-genome sequences of every isolate using novel nanopore protocols. FINDINGS: 118 genetically linked poliovirus isolates related to the serotype 2 Sabin vaccine strain were detected in 21 of 52 sequential sewage samples collected in London between Feb 8 and July 4, 2022. Expansion of environmental surveillance sites in London helped localise transmission to several boroughs in north and east London. All isolates have lost two key attenuating mutations, are recombinants with a species C enterovirus, and an increasing proportion (20 of 118) meet the criterion for a vaccine-derived poliovirus, having six to ten nucleotide changes in the gene coding for VP1 capsid protein. INTERPRETATION: Environmental surveillance allowed early detection of poliovirus importation and circulation in London, permitting a rapid public health response, including enhanced surveillance and an inactivated polio vaccine campaign among children aged 1-9 years. Whole-genome sequences generated through nanopore sequencing established linkage of isolates and confirmed transmission of a unique recombinant poliovirus lineage that has now been detected in Israel and the USA. FUNDING: Medicines and Healthcare products Regulatory Agency, UK Health Security Agency, Bill & Melinda Gates Foundation, and National Institute for Health Research Medical Research Council.

Postpandemic rebound of adeno-associated virus type 2 (AAV2) infections temporally associated with an outbreak of unexplained severe acute hepatitis in children in the United Kingdom.

Over 1000 cases of unexplained severe acute hepatitis in children have been reported to date worldwide. An association with adeno-associated virus type 2 (AAV2) infection, a human parvovirus, prompted us to investigate the epidemiology of AAV in the United Kingdom. Three hundred pediatric respiratory samples collected before (April 03, 2009-April 03, 2013) and during (April 03, 2022) the COVID-19 pandemic were obtained. Wastewater samples were collected from 50 locations in London (August 2021-March 2022). Samples were tested for AAV using real-time polymerase chain reaction followed by sequencing. Selected adenovirus (AdV)-positive samples were also sequenced. The detection frequency of AAV2 was a sevenfold higher in 2022 samples compared with 2009-2013 samples (10% vs. 1.4%) and highest in AdV-positive samples compared with negatives (10/37, 27% vs. 5/94, 5.3%, respectively). AAV2-positive samples displayed high genetic diversity. AAV2 sequences were either very low or absent in wastewater collected in 2021 but increased in January 2022 and peaked in March 2022. AAV2 was detected in children in association with AdV of species C, with a highest frequency in 2022. Our findings are consistent with the expansion of the population of children unexposed to AAV2, leading to greater spread of the virus once distancing restrictions were lifted.

Dynamic population normalisation in wastewater-based epidemiology for improved understanding of the SARS-CoV-2 prevalence: a multi-site study.

Wastewater-based epidemiology (WBE) is a valuable tool for monitoring the circulation of COVID-19. However, while variations in population size are recognised as major sources of uncertainty, wastewater SARS-CoV-2 measurements are not routinely population-normalised. This paper aims to determine whether dynamic population normalisation significantly alters SARS-CoV-2 dynamics observed through wastewater monitoring, and whether it is beneficial or necessary to provide an understanding of COVID-19 epidemiology. Data from 394 sites in England are used, and normalisation is implemented based on ammoniacal nitrogen and orthophosphate concentrations. Raw and normalised wastewater SARS-CoV-2 metrics are evaluated at the site and spatially aggregated levels are compared against indicators of prevalence based on the Coronavirus Infection Survey and Test and Trace polymerase chain reaction test results. Normalisation is shown, on average, to have a limited impact on overall temporal trends. However, significant variability in the degree to which it affects local-level trends is observed. This is not evident from previous WBE studies focused on single sites and, critically, demonstrates that while the impact of normalisation on SARS-CoV-2 trends is small on average, this may not always be the case. When averaged across many sites, normalisation strengthens the correlation between wastewater SARS-CoV-2 data and prevalence indicators; however, confidence in the improvement is low.

Research needs for optimising wastewater-based epidemiology monitoring for public health protection.

Wastewater-based epidemiology (WBE) is an unobtrusive method used to observe patterns in illicit drug use, poliovirus, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The pandemic and need for surveillance measures have led to the rapid acceleration of WBE research and development globally. With the infrastructure available to monitor SARS-CoV-2 from wastewater in 58 countries globally, there is potential to expand targets and applications for public health protection, such as other viral pathogens, antimicrobial resistance (AMR), pharmaceutical consumption, or exposure to chemical pollutants. Some applications have been explored in academic research but are not used to inform public health decision-making. 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.

Hybrid modelling of water resource recovery facilities: status and opportunities.

Mathematical modelling is an indispensable tool to support water resource recovery facility (WRRF) operators and engineers with the ambition of creating a truly circular economy and assuring a sustainable future. Despite the successful application of mechanistic models in the water sector, they show some important limitations and do not fully profit from the increasing digitalisation of systems and processes. Recent advances in data-driven methods have provided options for harnessing the power of Industry 4.0, but they are often limited by the lack of interpretability and extrapolation capabilities. Hybrid modelling (HM) combines these two modelling paradigms and aims to leverage both the rapidly increasing volumes of data collected, as well as the continued pursuit of greater process understanding. Despite the potential of HM in a sector that is undergoing a significant digital and cultural transformation, the application of hybrid models remains vague. This article presents an overview of HM methodologies applied to WRRFs and aims to stimulate the wider adoption and development of HM. We also highlight challenges and research needs for HM design and architecture, good modelling practice, data assurance, and software compatibility. HM is a paradigm for WRRF modelling to transition towards a more resource-efficient, resilient, and sustainable future.

Bifurcation Analysis of an Impulsive System Describing Partial Nitritation and Anammox in a Hybrid Reactor.

Low-energy nitrogen removal under mainstream conditions is a technology that has received significant attention in recent years as the water industry drives toward long-term sustainability goals. Simultaneous partial nitritation-Anammox (PN/A) is one process that can provide substantial energy reduction and lower sludge yields. Mathematical modeling of the PN/A process offers engineers insights into the operating conditions necessary to maximize its potential. Laureni et al. (Laureni et al. Water Res. 2019, 14) have recently published a simplified mechanistic model of the process operated as a sequencing batch reactor that investigated the effect of three key operating parameters on performance (Anammox biofilm activity, dissolved oxygen concentration and fraction of solids wasted). The analysis of the model was limited, however, to simulation with relatively few discrete parameter sets. Here, we demonstrate through the use of bifurcation theory applied to an impulsive dynamical system that the parameter space can be partitioned into regions in which the system converges to different fixed points that represent different outcomes: either the washout of nitrite-oxidizing bacteria or their survival. Mapping process performance data onto these spaces allows engineers to target suitable operating regimes for specific objectives. Here, for example, we note that the nitrogen removal efficiency is maximized close to the curve that separates the regions in parameter space where nitrite-oxidizing bacteria washout from the region in which they survive. Further, control of solids washout and Anammox biofilm activity can also reduce oxygen requirements while maintaining an appropriate hydraulic retention time. The approach taken is significant given the possibility for using such a methodology for models of increasing complexity. This will enable engineers to probe the entire parameter space of systems of higher dimension and realism in a consistent manner.

Color, structure, and rheology of a diblock bottlebrush copolymer solution.

A structure-property-process relation is established for a diblock bottlebrush copolymer solution, through a combination of rheo-neutron scattering, imaging, and rheological measurements. Polylactic acid-b-polystyrene diblock bottlebrush copolymers were dispersed in toluene with a concentration of 175 mg ml-1, where they self-assembled into a lamellar phase. All measurements were carried out at 5 °C. The solution color, as observed in reflection, is shown to be a function of the shear rate. Under equilibrium and near-equilibrium conditions, the solution has a green color. At low shear rates the solution remains green, while at intermediate rates the solution is cyan. At the highest rates applied the solution is indigo. The lamellar spacing is shown to be a decreasing function of shear rate, partially accounting for the color change. The lamellae are oriented 'face-on' with the wall under quiescence and low shear rates, while a switch to 'edge-on' is observed at the highest shear rates, where the reflected color disappears. The intramolecular distance between bottlebrush polymers does not change with shear rate, although at high shear rates, the bottlebrush polymers are preferentially aligned in the vorticity direction within the lamellae. We therefore form a consistent relation between structure and function, spanning a wide range of length scales and shear rates.

Identification of dropout predictors to a community-based physical activity programme that uses motivational interviewing.

BACKGROUND: Participant dropout reduces intervention effectiveness. Predicting dropout has been investigated for Exercise Referral Schemes, but not physical activity (PA) interventions with Motivational Interviewing (MI). METHODS: Data from attendees (n = 619) to a community-based PA programme utilizing MI techniques were analysed using a chi-squared test to determine dropout and attendance group differences. Binary logistic regression investigated the likelihood of dropout before 12 weeks. RESULTS: A total of 44.7% of participants dropped out, with statistical (P < 0.05) differences between groups for age, PA and disability. Regression for each variable showed participants aged 61-70 years (OR = 0.28, CI = 0.09-0.79; P = 0.018), >70 years (OR = 0.30, CI = 0.09-0.90; P = 0.036), and high PA (OR = 0.40, CI = 0.20-0.75; P = 0.006) reduced dropout likelihood. Endocrine system disorders (OR = 4.24, CI = 1.19-19.43; P = 0.036) and musculoskeletal disorders (OR = 3.14, CI = 1.84-5.45; P < 0.001) increased dropout likelihood. Significant variables were combined in a single regression model. Dropout significantly reduced for 61-70 years old (OR = 0.31, CI = 0.10-0.90; P = 0.035), and high PA (OR = 0.39, CI = 0.19-0.76; P = 0.008). Musculoskeletal disorders increased dropout (OR = 2.67, CI = 1.53-4.75; P < 0.001). CONCLUSIONS: Age, PA and disability type significantly influence dropout at 12 weeks. These are the first results specific to MI based programmes indicating the inclusion of MI and highlighting the need for further research.

Dilute solution structure of bottlebrush polymers.

Bottlebrush polymers are a class of macromolecules that have recently found use in a wide variety of materials, ranging from lubricating brushes and nanostructured coatings to elastomeric gels that exhibit structural colors. These polymers are characterized by dense branches extending from a central backbone and thus have properties distinct from linear polymers. It remains a challenge to specifically understand conformational properties of these molecules, due to the wide range of architectural parameters that can be present in a system, and thus there is a need to accurately characterize and model these molecules. In this paper, we use a combination of viscometry, light scattering, and computer simulations to gain insight into the conformational properties of dilute solution bottlebrush polymers. We focus on a series of model bottlebrushes consisting of a poly(norbornene) (PNB) backbone with poly(lactic acid) (PLA) side chains. We demonstrate that intrinsic viscosity and hydrodynamic radius are experimental observations sensitive to molecular architecture, exhibiting distinct differences with different choices of branches and backbone lengths. Informed by the atomistic structure of this PNB-PLA system, we rationalize a coarse-grained simulation model that we evaluate using a combination of Brownian dynamics and Monte Carlo simulations. We show that this exhibits quantitative matching to experimental results, enabling us to characterize the overall shape of the bottlebrush via a number of metrics that can be extended to more general bottlebrush architectures.

Programming and supervision of resistance training leads to positive effects on strength and body composition: results from two randomised trials of community fitness programmes.

BACKGROUND: Many sedentary adults have high body fat along with low fitness, strength, and lean body mass (LBM) which are associated with poor health independently of body mass. Physical activity can aid in prevention, management, and treatment of numerous chronic conditions. The potential efficacy of resistance training (RT) in modifying risk factors for cardiovascular and metabolic disease is clear. However, RT is under researched in public health. We report community-based studies of RT in sedentary (Study 1), and overweight and pre-diabetic (Study 2) populations. METHODS: Study 1 - A semi randomised trial design (48-weeks): Participants choosing either a fitness centre approach, and randomised to structured-exercise (STRUC, n = 107), or free/unstructured gym use (FREE, n = 110), or not, and randomised to physical-activity-counselling (PAC, n = 71) or a measurement only comparator (CONT, n = 76). Study 2 - A randomised wait list controlled trial (12-weeks): Patients were randomly assigned to; traditional-supervised-exercise (STRUC, n = 30), physical-activity-counselling (PAC, n = 23), either combined (COMB, n = 39), or a wait-list comparator (CONT, n = 54). Outcomes for both were BF mass (kg), LBM (kg), BF percentage (%), and strength. RESULTS: Study 1: One-way ANCOVA revealed significant between group effects for BF% and LBM, but not for BF mass or strength. Post hoc paired comparisons revealed significantly greater change in LBM for the STRUC group compared with the CONT group. Within group changes using 95%CIs revealed significant changes only in the STRUC group for both BF% (- 4.1 to - 0.9%) and LBM (0.1 to 4.5 kg), and in FREE (8.2 to 28.5 kg) and STRUC (5.9 to 26.0 kg) for strength. Study 2: One-way ANCOVA did not reveal significant between group effects for strength, BF%, BF mass, or LBM. For strength, 95%CIs revealed significant within group changes for the STRUC (2.4 to 14.1 kg) and COMB (3.7 to 15.0 kg) groups. CONCLUSION: Strength increased in both studies across all RT treatments compared to controls, yet significant improvements in both strength and body-composition occurred only in programmed and/or supervised RT. As general increases in physical activity have limited impact upon body-composition, public health practitioners should structure interventions to include progressive RT. TRIAL REGISTRATION: Study 1: ISRCTN13024854 , retrospectively registered 20/02/2018. Study 2: ISRCTN13509468 , retrospectively registered 20/02/2018).

Primary vs secondary adrenal insufficiency: ACTH-stimulated aldosterone diagnostic cut-off values by tandem mass spectrometry.

OBJECTIVES: To validate the diagnostic utility of Cortrosyn(™) stimulated aldosterone in the differentiation of primary (PAI) and secondary adrenal insufficiency (SAI) and to evaluate the effect of urine sodium levels and posture on test performance. DESIGN: Cross-sectional study. METHODS: Healthy volunteers (HV; n = 46) and patients with PAI (n = 26) and SAI (n = 29) participated in the study. Testing included cortisol and aldosterone (by liquid-chromatography tandem mass spectrometry) measurements at baseline and 30 and 60 min after 250 µg Cortrosyn(™). Plasma corticotropin (ACTH), renin activity (PRA) and urine spot sodium as a proxy for 24-h urine sodium excretion were measured at baseline. The effect of a sitting or semifowlers posture was evaluated in healthy volunteers. RESULTS: A Cortrosyn(™)-stimulated aldosterone level of 5 ng/dl (0·14 nmol/l) had 88% sensitivity and positive predictive value and 89·7% specificity and negative predictive value for distinguishing PAI from SAI. Spot urine sodium levels showed a strong correlation with peak aldosterone levels (r = -0·55, P = 0·02, n = 18) in the SAI but not PAI or HV groups. Posture did not have a significant effect on results. CONCLUSIONS: Once diagnosed with adrenal insufficiency, a stimulated aldosterone value of 5 ng/dl (0·14 nmol/l) works well to differentiate PAI from SAI. However, clinicians should be aware of the possible effect of total body sodium as reflected by spot urine sodium levels on aldosterone results. A 24-h urine sodium measurement may be helpful in interpretation.

Results of toric intraocular lenses for post-penetrating keratoplasty astigmatism.

PURPOSE: Evaluate the usefulness of toric intraocular lens (IOL) implantation during cataract surgery in patients after penetrating keratoplasty (PKP). DESIGN: Retrospective case review. PARTICIPANTS: A total of 21 eyes of 16 patients with prior PKP and moderate to high regular astigmatism after full suture removal underwent phacoemulsification and implantation of a single-piece acrylic toric IOL (SN6AT series; Alcon, Fort Worth, TX). METHODS: Patients underwent comprehensive examinations at standard intervals, including visual acuity, manifest refraction, and corneal topography. MAIN OUTCOME MEASURES: Uncorrected distance visual acuity (UDVA) and corrected distance visual acuity (CDVA) expressed as the logarithm of the minimum angle of resolution (logMAR) and manifest refraction astigmatism. RESULTS: From preoperatively to the last visit (mean, 14.7 ± standard deviation 12.8 months), the 21 eyes had significant improvement in UDVA (logMAR, 0.90 ± 0.48 to 0.23 ± 0.25; P = 0.0001) and CDVA (logMAR, 0.31 ± 0.14 to 0.08 ± 0.13; P = 0.0001). A total of 14 of 21 eyes (67%) and 17 of 21 eyes (81%) had UDVA and CDVA of ≥ 20/30, respectively. Preoperative topographic astigmatism was 4.57 ± 2.05 diopters (D). Postoperative manifest refraction astigmatism was 1.58 ± 1.25 D overall, but lower (0.75 ± 0.54 D) in the T7-T9 subgroup (excluding 1 outlier whose corneal astigmatism doubled after surgery) than in the T4-T6 subgroup (1.88 ± 1.28 D; P = 0.013). A total of 16 of all 21 eyes (76.2%) and 8 of 9 eyes (89%) in the T7-T9 subgroup were within 1 D of postoperative manifest astigmatism as predicted or better. CONCLUSIONS: Toric IOLs placed during cataract surgery after PKP and full suture removal can reduce manifest refraction cylinder to predictably low levels with corresponding improvement in UDVA and CDVA in patients with moderate to high regular preoperative topographic astigmatism.

Piloting wastewater-based surveillance of norovirus in England.

Wastewater-based epidemiology (WBE) gained widespread use as a tool for supporting clinical disease surveillance during the COVID-19 pandemic. There is now significant interest in the continued development of WBE for other pathogens of clinical significance. In this study, approximately 3,200 samples of wastewater from across England, previously collected for quantification of SARS-CoV-2, were re-analysed for the quantification of norovirus genogroup I (GI) and II (GII). Overall, GI and GII were detected in 93% and 98% of samples respectively, and at least one of the genogroups was detected in 99% of samples. GI was found at significantly lower concentrations than GII, but the proportion of each genogroup varied over time, with GI becoming more prevalent than GII in some areas towards the end of the study period (May 2021 - March 2022). Using relative strength indices (RSI), it was possible to study the trends of each genogroup, and total norovirus over time. Increases in norovirus levels appeared to coincide with the removal of COVID-19 related lockdown restrictions within England. Local Moran's I analyses indicated several localised outbreaks of both GI and GII across England, notably the possible GI outbreak in the north of England in early 2022. Comparisons of national average norovirus concentrations in wastewater against concomitant norovirus reported case numbers showed a significant linear relationship. This highlights the potential for wastewater-based monitoring of norovirus as a valuable approach to support surveillance of norovirus in communities.

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.

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.