Wastewater-based Epidemiology Research Articles

Towards Evidence-Based Food Safety Governance with Wastewater-Based Epidemiology (WBE) Technology in China

Towards Evidence-Based Food Safety Governance with Wastewater-Based Epidemiology (WBE) Technology in China

Submersible voltammetric sensing probe for rapid and extended remote monitoring of opioids in community water systems

The intensifying global opioid crisis, majorly attributed to fentanyl (FT) and its analogs, has necessitated the development of rapid and ultrasensitive remote/on-site FT sensing modalities. However, current approaches for tracking FT exposure through wastewater-based epidemiology (WBE) are unadaptable, time-consuming, and require trained professionals. Toward developing an extended in situ wastewater opioid monitoring system, we have developed a screen-printed electrochemical FT sensor and integrated it with a customized submersible remote sensing probe. The sensor composition and design have been optimized to address the challenges for extended in situ FT monitoring. Specifically, ZIF-8 metal–organic framework (MOF)-derived mesoporous carbon (MPC) nanoparticles (NPs) are incorporated in the screen-printed carbon electrode (SPCE) transducer to improve FT accumulation and its electrocatalytic oxidation. A rapid (10 s) and sensitive square wave voltammetric (SWV) FT detection down to 9.9 µgL−1 is thus achieved in aqueous buffer solution. A protective mixed-matrix membrane (MMM) has been optimized as the anti-fouling sensor coating to mitigate electrode passivation by FT oxidation products and enable long-term, intermittent FT monitoring. The unique MMM, comprising an insulating polyvinyl chloride (PVC) matrix and carboxyl-functionalized multi-walled carbon nanotubes (CNT-COOH) as semiconductive fillers, yielded highly stable FT sensor operation (> 95% normalized response) up to 10 h in domestic wastewater, and up to 4 h in untreated river water. This sensing platform enables wireless data acquisition on a smartphone via Bluetooth. Such effective remote operation of submersible opioid sensing probes could enable stricter surveillance of community water systems toward timely alerts, countermeasures, and legal enforcement.Graphical abstract

Graphene Multiplexed Sensor for Point-of-Need Viral Wastewater-Based Epidemiology.

Wastewater-based epidemiology (WBE) can help mitigate the spread of respiratory infections through the early detection of viruses, pathogens, and other biomarkers in human waste. The need for sample collection, shipping, and testing facilities drives up the cost of WBE and hinders its use for rapid detection and isolation in environments with small populations and in low-resource settings. Given the ubiquitousness and regular outbreaks of respiratory syncytial virus, SARS-CoV-2, and various influenza strains, there is a rising need for a low-cost and easy-to-use biosensing platform to detect these viruses locally before outbreaks can occur and monitor their progression. To this end, we have developed an easy-to-use, cost-effective, multiplexed platform able to detect viral loads in wastewater with several orders of magnitude lower limit of detection than that of mass spectrometry. This is enabled by wafer-scale production and aptamers preattached with linker molecules, producing 44 chips at once. Each chip can simultaneously detect four target analytes using 20 transistors segregated into four sets of five for each analyte to allow for immediate statistical analysis. We show our platform's ability to rapidly detect three virus proteins (SARS-CoV-2, RSV, and Influenza A) and a population normalization molecule (caffeine) in wastewater. Going forward, turning these devices into hand-held systems would enable wastewater epidemiology in low-resource settings and be instrumental for rapid, local outbreak prevention.

Amphetamine-like substances and synthetic cathinones in Portuguese wastewater influents: Enantiomeric profiling and role of suspended particulate matter

Wastewater based epidemiology (WBE) has been used worldwide to estimate drug consumption routinely. Even though WBE provides valuable data to support legal and health interventions associated to drug use, monitoring studies in Portuguese wastewaters are scarce. Hence, this work aimed to estimate the consumption of some conventional abuse and illicit drugs such as amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxymethamphetamine (MDMA), and the synthetic cathinones buphedrone (BPD), butylone (BTL), 3,4-dimethylmethcathinone (3,4-DMMC) and 3-methylmethcathinone (3-MMC), considering not only the liquid phase, but also the suspended particulate matter (SPM). Moreover, the enantiomeric profiling of the samples was studied, exploring for the first time the possible enantioselective sorption of these drugs onto SPM. For that, 24 h composite raw wastewaters were collected from a conventional wastewater treatment plant (WWTP) in Portugal. After extraction, the liquid phase and SPM extracts were derivatized with an enantiomerically pure reagent and then, analysed using a gas chromatography-mass spectrometry (GC-MS) analytical method. The results showed a low and non-enantioselective adsorption to SPM at environmental relevant levels. Only (S)-AMP was detected in two SPM samples, whereas AMP, MAMP, MDMA, BPD, and 3,4-DMMC were detected in the liquid phase. AMP was the most frequently found drug with an estimated load up to 166.0 mg day−1 1000 people−1 and mostly found with enrichment of (S)-AMP. Nevertheless, (R)-AMP was also determined, which may be related to the consumption of either the illicit racemic AMP or the medicine (R)-deprenyl. The use of MDMA, MAMP and synthetic cathinones (BPD and 3,4-DMMC) was also suggested in Portugal. Nevertheless, the levels and the consumption estimate of the target chemicals were lower than in other European countries or worldwide. These findings provide the first step to the implementation of WBE monitoring campaigns to assess the status of drug consumption in Portuguese communities, contributing to the understanding of drug use patterns and trends worldwide and helping enforce preventive measures.

Assessing daily patterns in stimulant use during the COVID-19 pandemic in Melbourne, Australia using wastewater analysis

During the COVID-19 pandemic, one of Australia’s biggest cities, Melbourne, experienced three major isolation (“lockdown”) periods in 2020 (160 days) and in 2021 (111 days) which makes it one of the most locked down cities world-wide. This study assessed how the pandemic affected temporal trends in methamphetamine, MDMA and cocaine consumption using wastewater-based epidemiology. Daily samples were collected for most of 2020 and 2021 (n = 660 days). Concentrations were measured using direct-injection LC-MS/MS and back-calculated to consumption estimates. Results indicate that methamphetamine use was increasing before the first lockdown and decreased after the end of the first lockdown in 2020. Methamphetamine trends appeared to have remained steady throughout the second lockdown period before increasing steeply after it ended. For most of 2020, cocaine use remained steady, with an increase after the second lockdown. MDMA use decreased after the start of the first lockdown and remained steady throughout most of 2020 and 2021. In comparison to 2020, trends in 2021 were less variable and stimulant use did not appear to be as associated with COVID-19 restrictions. Overall, this study was able to show the impact of lockdown periods and the related social restrictions on illicit stimulant use. Environmental ImplicationIllicit drugs are hazardous chemicals, of concern both to humans and the environment. While studies have been undertaken to understand their temporal trends, this work utilizes wastewater-based epidemiology and daily sampling to provide a comprehensive understanding of the impact of the COVID-19 pandemic on the use of methamphetamine, MDMA and cocaine on one of the most locked-down cities in the world. Understanding the consequences of this significant intervention on illicit drug use could provide valuable insights into its potential environmental impact.

Nationwide assessment of illicit drug consumption patterns in South Korea using wastewater-based epidemiology during the COVID-19 pandemic (2020–2022)

Illicit drugs have become a crucial global social issue, with South Korea experiencing a continuous increase in the number of offenders and drug smuggling. This study employed wastewater-based epidemiology to investigate consumption patterns of 8 illicit drugs and their 7 metabolites during the COVID-19 pandemic (2020–2022) in South Korea. Ten compouds were detected in the wastewater influent. Methamphetamine (METH) was prevalent in samples, followed by amphetamine and ecstasy (MDMA). Interestingly, MDMA and ketamine (KET), which were not detected in previous Korean studies conducted before COVID-19 pandemic, were detected in this study. METH exhibited the highest consumption rates, decreasing from 16.6 to 12.4 mg/day/1000 people between 2020 and 2022, while MDMA increased over the three years (mean: 1.16, 1.24, and 1.62 mg/day/1000 people in 2020, 2021, and 2022, respectively) (p < 0.05). Significant correlations were identified between regional income levels and the consumption rates of METH (p < 0.01), MDMA (p < 0.01), and KET (p < 0.05). Furthermore, METH and MDMA consumption rates in cities were positively correlated with the number of drug offenders arrested and local clubs in those cities. The findings of this study provide valuable insights into shaping regulatory policies related to illicit drugs and future studies.

Hepatitis A in Latin America: The current scenario.

This review aims to gather and disseminate updated information regarding hepatitis A virus (HAV) in Latin America (LA) in the last 11years, including seroprevalence, post-vaccination studies, virus detection in aqueous matrices and food samples, and outbreak reports. Only 24 seroprevalence studies were published between 2012 and 2023 with 55%-100% reported prevalences of anti-HAV IgG. Among the 25 LA countries, only eight of them have introduced HAV vaccines into their immunisation programs. Outbreaks of hepatitis A occurred between 2017-2019, mainly affecting men who have sex with men in Argentina, Brazil and Chile, probably as a consequence of the abrupt decline of young adults' immunity. This could be due to that young adult have never been infected in childhood (due to socio-health improvements) and are above the cut-off ages to be included when the vaccination programs were introduced. Although scarce, studies focused on environmental and food HAV surveillance have shown viral presence in these samples. Surface waters presented HAV detections between 1.2% and 86.7%, and untreated wastewaters between 2.8% and 70.9%. Genotypes found in all cases were IA and IC. The only wastewater-based epidemiology study showed to be a useful tool as a complement of traditional epidemiological surveillance. Only four LA countries have looked for HAV in food samples, with genome detection rates between 9% and 33%. Latin American HAV circulation scenario is changing. In countries where socioeconomic and sanitary conditions have not improved, the virus persists with high endemicity and the access to the vaccine should be re-evaluated by local governments. In countries where access to clean water, better sanitary conditions and HAV immunisation programs have been implemented, the number of cases among young adults seems to be increasing, alerting health authorities.

Integrated hepatitis e virus monitoring in central Argentina: a six-year analysis of clinical surveillance and wastewater-based epidemiology

Wastewater-based epidemiology (WBE) has gained prominence worldwide as a powerful tool in public health. This study aimed to monitor the circulation of Hepatitis E Virus (HEV) from wastewater samples collected during a six-year period and compare these results with clinical surveillance in the central region of Argentina. From 2017 to 2022, 1008 raw wastewater samples were analyzed, including four wastewater treatment plants from four cities (n=319), and 7 local neighborhood collector sewers in Córdoba city (n=689). Serum and/or stool samples from patients suspected of HEV infection were also analyzed (n=48). HEV molecular detection and viral load quantification were performed by real time RT-qPCR, and genetic characterization by two RT-Nested PCRs (targeting partial ORF-1 and ORF-2 genomic regions), sequencing and phylogenetic analysis.Fifty-three (5.3%) wastewater samples were RNA-HEV positive by real time RT-qPCR, with variations according to the location and year (0.0% - 21.6%). Out of these, ORF-2 genomic region was amplified in 20 samples (37.7%) and ORF-1 partial region in 12 (22.6%), and eighteen sequences were obtained. Throughout the study period, two (4.2%) HEV confirmed infections were reported, and one sequence was obtained. Phylogenetic analyses for both genomic regions showed that all the isolates were genotype HEV-3 clade abchijklm.Our study detected HEV in wastewater over a six-year period, despite a low number of clinical cases, emphasizing WBE as a valuable tool that complements clinical surveillance, by detecting pathogens' presence; identifying their transmission, circulation dynamics and excretion hotspots; and revealing changes in their genomic diversity.

Evaluating storage conditions and enhancement strategies on viral biomarker recovery for WBE applications.

Wastewater-based epidemiology (WBE) is a valuable disease surveillance tool. However, little is known on how factors such as transportation, storage, and wastewater characteristics influence the accuracy of the quantification methods. Hence, this study investigated the impact of storage temperatures and physicochemical characteristics of wastewater on SARS-CoV-2 and influenza A stability using droplet digital PCR. Additionally, strategies to enhance viral recovery were explored. Municipal influent wastewater stored between ±25 and -80 °C was assessed for a period of 84 days to determine viral degradation. Degradation up to 94.1% of influenza A and SARS-CoV-2 was observed in all samples with the highest at ±25 °C. Viral degradation was correlated to the changes in wastewater physicochemical characteristics. The low degradation observed of SARS-CoV-2 in the spiked pellets were indicative of viral adhesion to wastewater solids, which correlated with changes in pH. Ultrasonication frequencies ranging from 4 to 16 kHz, increased SARS-CoV-2 concentrations in the supernatant between 3.30 and 35.65%, indicating viral RNA attachment to wastewater solids. These results highlight the importance of additional pretreatment methods for maximizing RNA recovery from wastewater samples. Based on these findings, it was deduced that wastewater preservation studies are essential, and pretreatment should be included in the WBE methodology.

Dilute-and-shoot approach for the high-throughput LC-MS/MS determination of illicit drugs in the field of wastewater-based epidemiology

The determination of illicit drugs in urban influent wastewater (IWW) enables the monitoring of spatial and temporal drug usage trends and assessment of community lifestyle habits. The increasing number of wastewater surveillance studies has emphasized the necessity for the development of rapid, high-throughput methods that maintain high quality data. This work evaluates the use of a dilute-and-shoot methodology, based on direct injection (DI) of centrifuged samples, as an alternative approach to the widely applied sample pre-treatment based on solid-phase extraction, for the liquid chromatography-tandem mass spectrometry determination of seven widely consumed illicit drugs and their metabolites in IWW (amphetamine; cocaine metabolite, benzoylecgonine; ketamine; 3,4-methylenedioxymethamphetamine (MDMA); methamphetamine; cannabis metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH); heroin metabolite, 6-acetylmorphine (6-MAM)). Comparison of both approaches in terms of matrix effects, sensitivity and accuracy, demonstrates the DI method suitability to correctly quantify these analytes in IWW, with a limit of quantification lower than 30 ng L−1 for most compounds. After validation of the method and participation in an interlaboratory exercise, the DI method was applied to the analysis of 54 IWW samples collected from different Spanish wastewater treatment plants. Additionally, quality controls were incorporated in each analysis batch to support the DI method applicability and robustness. The use of a 10 μL-DI reduces time-consuming sample preparation, analysis time and measurement uncertainty. Moreover, it supports green chemistry by reducing the consumption of organic solvents and it facilitates logistics by collecting, transporting, and storing less sample volume. The methodology is therefore especially appropriate for monitoring illicit drugs in large wastewater-based epidemiology sampling campaigns or when fast near real-time results are needed.

Urban Wastewater-Based Surveillance of SARS-CoV-2 Virus: A Two-Year Study Conducted in City of Patras, Greece.

Wastewater-based epidemiology, during the COVID-19 pandemic years, has been applied as a complementary approach, worldwide, for tracking SARS-CoV-2 virus into the community and used as an early warning of the prevalence of COVID-19 infection. The present study presents the results of the 2-year surveillance project, in the city of Patras, Greece. The purpose of the study was to monitor SARS-CoV-2 and implement WBE as an early warning method of monitoring Public Health impact. The presence of SARS-CoV-2 was determined and quantified in 310 samples using RT-qPCR assays. For the years 2022 and 2023, 93.5% and 78.7% of samples were found positive, respectively. Comparison of detection methods have been conducted to select the method with the highest recovery of the viral load. A seasonal variation of the virus was recorded, showing a recession in summer months confirming the country's epidemiological data as indicated by positive correlation of wastewater viral load with registered cases of COVID-19 infections during these years (p < 0.05) and moreover sealed with a significant negative correlation observed with Daily Average (p < 0.01) and Daily Maximum Temperature (p < 0.01). More research was carried out to elucidate a possible association of physicochemical characteristics of wastewater with viral load showing positive correlation with Chlorides (p < 0.01) advocating possible increased use of chlorine-based disinfectants and Electrical Conductivity (p < 0.01) indicates that wastewater during periods of increased infections is more heavily loaded with ions from chemical and biological pollutants. No correlation found with rainfall and physicochemical indicators, such as COD, BOD5, Total Phosphorus, Total Nitrogen, and Total Suspended Solids. According to the findings, WBE represents a useful tool in the management of epidemics based on an environmental approach and it can also shed light on the interacting parameters that capture Public Health since any infections that may lead to epidemics lead to a parallel change in the use of pharmaceuticals, antimicrobials, disinfectants, and microbial load in urban wastewater.

Feasibility of Wastewater‐based Public Health Monitoring Systems in Texas’ Small Rural Communities

AbstractIn recent years, there has been much focus on the use of wastewater‐based epidemiology (WBE) in urban centers, particularly for SARS‐CoV‐2 monitoring. However, less is known about the application of WBE in rural settings or in areas of limited resources. Most WBE programs in low‐resource communities have occurred outside the United States. To reap the benefits, WBE would need to be tailored to better reflect the socioeconomic challenges, technical barriers, communication limitations, and variable wastewater infrastructures associated with rural communities. The objective of this review is to evaluate the potential opportunities and challenges of deploying the current SARS‐CoV‐2 monitoring methodologies in small, rural communities, with a particular focus on rural Texas. For this, we conducted an inventory of rural communities in the state of Texas and their wastewater infrastructure. Based on specific rural examples, we evaluated the potential of current WBE methodologies used in urban settings to monitor for emerging biological agents of concern such as SARS‐CoV‐2. Our findings include an overview of rural wastewater capacity across rural Texas, a look at current WBE efforts to detect SARS‐CoV‐2, and recommendations for future implementation in two cities in rural counties, Kerrville and Valentine. WBE is a rapidly evolving public health tool with several notable advantages associated with cost, access, and adaptability. It is of particular use in resource‐limited communities that often exhibit healthcare disparities. This study presents the first overview of the feasibility of implementing WBE in the rural settings of Texas. We provide several recommendations and suggest alternatives that may be of use when planning an expansion of WBE into these areas.

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater

Population infection estimation from wastewater surveillance for SARS-CoV-2 in Nagpur, India during the second pandemic wave.

Wastewater-based epidemiology (WBE) has emerged as an effective environmental surveillance tool for predicting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease outbreaks in high-income countries (HICs) with centralized sewage infrastructure. However, few studies have applied WBE alongside epidemic disease modelling to estimate the prevalence of SARS-CoV-2 in low-resource settings. This study aimed to explore the feasibility of collecting untreated wastewater samples from rural and urban catchment areas of Nagpur district, to detect and quantify SARS-CoV-2 using real-time qPCR, to compare geographic differences in viral loads, and to integrate the wastewater data into a modified Susceptible-Exposed-Infectious-Confirmed Positives-Recovered (SEIPR) model. Of the 983 wastewater samples analyzed for SARS-CoV-2 RNA, we detected significantly higher sample positivity rates, 43.7% (95% confidence interval (CI) 40.1, 47.4) and 30.4% (95% CI 24.66, 36.66), and higher viral loads for the urban compared with rural samples, respectively. The Basic reproductive number, R0, positively correlated with population density and negatively correlated with humidity, a proxy for rainfall and dilution of waste in the sewers. The SEIPR model estimated the rate of unreported coronavirus disease 2019 (COVID-19) cases at the start of the wave as 13.97 [95% CI (10.17, 17.0)] times that of confirmed cases, representing a material difference in cases and healthcare resource burden. Wastewater surveillance might prove to be a more reliable way to prepare for surges in COVID-19 cases during future waves for authorities.

Long-term wastewater monitoring of SARS-CoV-2 viral loads and variants at the major international passenger hub Amsterdam Schiphol Airport: A valuable addition to COVID-19 surveillance

Wastewater-based epidemiological surveillance at municipal wastewater treatment plants has proven to play an important role in COVID-19 surveillance. Considering international passenger hubs contribute extensively to global transmission of viruses, wastewater surveillance at this type of location may be of added value as well. The aim of this study is to explore the potential of long-term wastewater surveillance at a large passenger hub as an additional tool for public health surveillance during different stages of a pandemic. Here, we present an analysis of SARS-CoV-2 viral loads in airport wastewater by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) from the beginning of the COVID-19 pandemic in Feb 2020, and an analysis of SARS-CoV-2 variants by whole-genome next-generation sequencing from Sep 2020, both until Sep 2022, in the Netherlands. Results are contextualized using (inter)national measures and data sources such as passenger numbers, clinical surveillance data and national wastewater surveillance data. Our findings show that wastewater surveillance was possible throughout the study period, irrespective of measures, as viral loads were detected and quantified in 98.6 % (273/277) of samples. Emergence of SARS-CoV-2 variants, identified in 91.0 % (161/177) of sequenced samples, coincided with increases in viral loads. Furthermore, trends in viral load and variant detection in airport wastewater closely followed, and in some cases preceded, trends in national daily average viral load in wastewater and variants detected in clinical surveillance. Wastewater-based epidemiology at a large international airport is a valuable addition to classical COVID-19 surveillance and the developed expertise can be applied in pandemic preparedness plans for other (emerging) pathogens in the future.

Insights into the panorama of multiple DNA viruses in municipal wastewater and recycled sludge in Tianjin, China

Environmental viruses in wastewater and sludge are widely recognized for their roles in waterborne diseases. However, previous studies mainly focused on RNA viruses, and little is known about the diversity of DNA viral communities and their driving factors in municipal wastewater treatment environments. Herein, we conducted a pilot study to explore DNA virus profiles in municipal wastewater and recycled sludge by metagenomics method, and track their temporal changes in northern China. Results showed that 467 viral species were co-shared among all the samples. We identified six families of human viruses with a prevalence of 0.1%, which were rare but relatively stable in wastewater and sludge for six months. Adenoviridae, Parvoviridae, and Herpersviridae were the most dominant human viral families in municipal wastewater and recycled sludge. A time series of samples revealed that the dynamic changes of human DNA viruses were stable based on qPCR results, particularly for high-risk fecal-oral transmission viruses of adenovirus, bocavirus, polyomavirus, human gamma herpesvirus, human papillomavirus, and hepatitis B virus. Concentrations of Adenovirus (5.39–7.48 log10 copies/L) and bocavirus (4.36–7.48 log10 copies/L) were observed to be the highest in these samples compared to other viruses. Our findings demonstrated the DNA viruses’ high prevalence and persistence in municipal wastewater treatment environments, highlighting the value of enhancing public health responses based on wastewater-based epidemiology.

Online dashboards for SARS-CoV-2 wastewater-based epidemiology.

Aim: Wastewater-based epidemiology (WBE) is increasingly used to monitor pandemics. In this manuscript, we review methods and limitations of WBE, as well as their online dashboards. Materials & methods: Online dashboards were retrieved using PubMed and search engines, and annotated for timeliness, availability of English version, details on SARS-CoV-2 sublineages, normalization by population and PPMoV load, availability of case/hospitalization count charts and of raw data for export. Results: We retrieved 51 web portals, half of them from Europe. Africa is represented from South Africa only, and only seven portals are available from Asia. Conclusion: WBS provides near-real-time cost-effective monitoring of analytes across space and time in populations. However, tremendous heterogeneity still persists in the SARS-CoV-2 WBE literature.

Monitoring an Emergent Pathogen at Low Incidence in Wastewater Using qPCR: Mpox in Switzerland.

Wastewater-based epidemiology offers a complementary approach to clinical case-based surveillance of emergent diseases and can help identify regions with infected people to prioritize clinical surveillance strategies. However, tracking emergent diseases in wastewater requires reliance on novel testing assays with uncertain sensitivity and specificity. Limited pathogen shedding may cause detection to be below the limit of quantification or bordering the limit of detection. Here, we investigated how the definition of limit of detection for quantitative polymerase chain reaction (qPCR) impacts epidemiological insights during an mpox outbreak in Switzerland. 365 wastewater samples from three wastewater treatment plants in Switzerland from 9 March through 31 October 2022 were analyzed for mpox DNA using qPCR. We detected mpox DNA in 22% (79 of 365) wastewater samples based on a liberal definition of qPCR detection as any exponentially increasing fluorescence above the threshold. Based on a more restrictive definition as the lowest concentration at which there is 95% likelihood of detection, detection was 1% (5 of 365). The liberal definition shows high specificity (90%) and accuracy (78%), but moderate sensitivity (64%) when benchmarked against available clinical case reporting, which contrasts with higher specificity (98%) but lower sensitivity (10%) and accuracy (56%) of the 95% likelihood definition. Wastewater-based epidemiology applied to an emergent pathogen will require optimizing public health trade-offs between reporting data with high degrees of uncertainty and delaying communication and associated action. Information sharing with relevant public health stakeholders could couple early results with clear descriptions of uncertainty.Impact Statement: When a novel pathogen threatens to enter a community, wastewater-based epidemiology offers an opportunity to track its emergence and spread. However, rapid deployment of methods for to detect a novel pathogen may rely on assays with uncertain sensitivity and specificity. Benchmarking the detection of mpox DNA in Swiss wastewaters with reported clinical cases in 2022, we demonstrate how definitions of detection of a qPCR assay influence epidemiological insights from wastewater. The results highlight the need for information sharing between public health stakeholders that couple early insights from wastewater with descriptions of methodological uncertainty to optimize public health actions.

Evaluation of different molecular systems for detection and quantification of SARS-CoV-2 RNA from wastewater samples

Wastewater-based epidemiology has proved to be a suitable approach for tracking the spread of epidemic agents including SARS-CoV-2 RNA. Different protocols have been developed for quantitative detection of SARS-CoV-2 RNA from wastewater samples, but little is known on their performance. In this study we compared three protocols based on Reverse Transcription Real Time-PCR (RT-PCR) and one based on Droplet Digital PCR (ddPCR) for SARS-CoV-2 RNA detection from 35 wastewater samples. Overall, SARS-CoV-2 RNA was detected by at least one method in 85.7 % of samples, while 51.4 %, 22.8 % and 8.6 % resulted positive with two, three or all four methods, respectively. Protocols based on commercial RT-PCR assays and on Droplet Digital PCR showed an overall higher sensitivity vs. an in-house assay. The use of more than one system, targeting different genes, could be helpful to increase detection sensitivity.

Cold-chain free nucleic acid preservation using porous super-absorbent polymer (PSAP) beads to facilitate wastewater surveillance

The instability of viral targets including SARS-CoV-2 in sewage is an important challenge in wastewater monitoring projects. The unrecognized interruptions in the ‘cold-chain’ transport from the sample collection to RNA quantification in the laboratory may undermine the accurate quantification of the virus. In this study, bovine serum albumin (BSA)-modified porous superabsorbent polymer (PSAP) beads were applied to absorb raw sewage samples as a simple method for viral RNA preservation. The preservation efficiency for SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA were examined during storage for 14 days at 4 °C or room temperature against the control (no beads applied). While a non-significant difference was observed at 4 °C (∼80 % retention for both control and PSAP-treated sewage), the reduction of SARS-CoV-2 RNA concentrations was significantly lower in sewage retrieved from PSAP beads (25–40 % reduction) compared to control (>60 % reduction) at room temperature. On the other hand, the recovery of PMMoV, known for its high persistence in raw sewage, from PSAP beads or controls were consistently above 85 %, regardless of the storage temperature. Our results demonstrate the applicability of PSAP beads to wastewater-based epidemiology (WBE) projects for preservation of SARS-CoV-2 RNA in sewage, especially in remote settings with no refrigeration capabilities.