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Wastewater-based epidemiology (WBE) estimates collective consumption or exposure to chemicals or pathogens by monitoring the substances excreted in the population’s wastewater. Advances in mass spectrometry (MS) and the application of some clinical diagnostic tools and proteomics to wastewater fingerprinting have been linked to the discovery of new biomarkers and indicators of population health and are broadening the scope of WBE that nowadays cover not only small molecule biomarkers but also genetic biomarkers, large molecules, viruses, infection diseases, resistance, etc. This mini-review highlights recent WBE advances using MS and how this progress can create a fingerprint of a city’s health hazards, habits, and lifestyle, which is gaining in public health emphasis., This work has been supported by Grant RTI2018-097158-BC31 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”, Grant PID2020-114065RBC22 funded by MCIN/ AEI/ 10.13039/501100011033 and project ANTROPOCEN@ (PROMETEO/2018/155) funded by Generalitat Valenciana.

Wastewater-based epidemiology (WBE) seeks to use biological or chemical indicators in sewage to provide information on the overall health of a community. This paper provides an overview of the range of applications of WBE over the past two decades, how it has been used to inform public health responses, and considerations for more integrated approaches to WBE based on a review of the literature. The review finds that WBE has been used extensively around the world for the estimation of consumption patterns of illicit drugs and other substances, but a range of novel applications also exist. As a result of the COVID-19 pandemic, many communities used WBE for the first time as a complementary public health surveillance tool, monitoring trends in SARS-CoV-2 prevalence in large cities, and for micro-surveillance on a more targeted level. WBE may continue to be a useful public health surveillance tool in the future; however, several limitations and challenges exist. Consideration of how information obtained through WBE can be used to inform public health responses is essential to understanding the potential costs and benefits compared with conventional public health surveillance techniques.

SARS-CoV-2 is shed by COVID-19 patients and can be detected in wastewater. Thus, testing wastewater for the virus provides a depiction of disease prevalence in a community. Virus concentration data can be utilized to monitor infection trends, identify hot spots, and inform decision makers regarding reopening efforts and directing resources. This perspective aims to shed light on the current situation relating to SARS-CoV-2 in the wastewater system and the opportunity to utilize wastewater to collect useful epidemiological data. First, the survivability of SARS-CoV-2 in different water matrices is examined through the lens of surrogate viruses. Second, the effect of wastewater treatment processes on SARS-CoV-2 is investigated. Current standards for sufficient reduction of the virus and the risk of exposure that arises at each stage in the wastewater treatment process are discussed. Third, the immense potential of wastewater-based epidemiology (WBE) for managing the ongoing COVID-19 pandemic is analyzed. Studies that have tested wastewater or sludge for SARS-CoV-2 are discussed, and results are tabulated. Lastly, the current limitations of WBE and opportunities of future research are explored. Using the wealth of knowledge that the scientific community now has about WBE, wastewater testing should be considered by regional governments and private institutions.

Graphical abstract An isotope pattern deconvolution (IPD) quantification method has been applied for the determination of five substances (amphetamine, benzoylecgonine, cocaine, methamphetamine and MDMA) in wastewater for the application in wastewater-based epidemiology (WBE). A previously validated method that used a calibration curve for quantification was modified to apply IPD. The two approaches were compared in terms of analytical uncertainty in recovery studies of quality control samples, i.e. six wastewater samples from different geographical origins spiked at two concentration levels. Both methods were reliable as they passed (z-score

Communities around the world have embraced the trend of wastewater monitoring to determine whether COVID-19 is circulating. From the start, the approach promised an inexpensive way to identify an o...

The correction factor (CF) of the alcohol biomarker ethyl sulfate (EtS), an important parameter for back-estimating alcohol consumption via wastewater-based epidemiology (WBE), reportedly has a large uncertainty due to the limited size of relevant pharmacokinetic studies. We applied an alternative approach to derive an EtS CF by comparing the sales data with the total load measured by WBE across Australia. We utilized EtS data from the National Wastewater Drug Monitoring Program in Australia from 2016 to 2019, that covered more than 50% of the Australian population. National sales data for alcohol were obtained from the Australian Bureau of Statistics from 2016 to 2018 - the most recent data available. A new CF of 4 × 103 was suggested for EtS, significantly greater than the CFs that have been used previously (3042 or 3320). This new CF was applied to a separate multiple-year set of WBE data for EtS from an Australian town and generated estimates that better match sales data than the previous CFs. The new CF incorporates both the excretion rate and sewer degradation of EtS and hence is likely to improve the future accuracy of alcohol consumption estimates by WBE.

New psychoactive substances (NPS), often designed as (legal) substitutes to conventional illicit drugs, are constantly emerging in the drug market and being commercialized in different ways and forms. Their use continues to cause public health problems and is therefore of major concern in many countries. Monitoring NPS use, however, is arduous and different sources of information are required to get more insight of the prevalence and diffusion of NPS use. The determination of NPS in pooled urine and wastewater has shown great potential, adding a different and complementary light on this issue. However, it also presents analytical challenges and limitations that must be taken into account such as the complexity of the matrices, the high sensitivity and selectivity required in the analytical methods as a consequence of the low analyte concentrations as well as the rapid transience of NPS on the drug market creating a scenario with constantly moving analytical targets. Analytical investigation of NPS in pooled urine and wastewater is based on liquid chromatography hyphenated to mass spectrometry and can follow different strategies: target, suspect and non-target analysis. This work aims to discuss the advantages and disadvantages of the different data acquisition workflows and data exploration approaches in mass spectrometry, but also pays attention to new developments such as ion mobility and the use of in-silico prediction tools to improve the identification capabilities in high-complex samples. This tutorial gives an insight into this emerging topic of current concern, and describes the experience gathered within different collaborations and projects supported by key research articles and illustrative practical examples. Refereed/Peer-reviewed

Illicit drug use in rural and suburban areas of China has not been studied extensively, as most studies have focused on illicit drug use in urban areas. To compare the differences between urban, suburban, and rural drug use, we collected influent samples from 19 urban, 9 suburban, and 18 rural wastewater treatment plants in Dalian, respectively. A method using solid-phase extraction combined with derivatization for gas chromatography -mass spectrometry analysis was applied to detect biomarker concentrations. The concentrations of methamphetamine and morphine ranged from 3.12 to 605 ng/L and

Alcohol and tobacco are the most frequently consumed substances in the world. Both are significantly associated with the increasing number of different diseases. Thus, monitoring nicotine and alcohol use is vital for public health planning and intervention strategies. This study aimed to calculate estimates of alcohol and nicotine use in 11 cities of Turkey using wastewater-based epidemiology. In 2019, daily composite wastewater samples from 18 wastewater treatment plants were collected for a week per season. The 24-h composite samples were collected via auto-samplers. Sample preparation for wastewater samples collected was done using liquid-liquid extraction and solid-phase extraction. Nicotine and ethyl sulfate (EtS) were analyzed using validated liquid chromatography-tandem mass spectrometry method. The estimated average nicotine consumption was 2.84 mg/p/day, and the average alcohol consumption was 3.46 ± 1.83 ml/p/day. The highest nicotine consumption was observed in Kayseri city; the highest alcohol consumption was calculated for Mersin city. In this study, the cigarette and alcohol consumption estimate obtained by wastewater-based epidemiology (WBE) was found to be higher than the Turkey Tobacco and Alcohol Market Regulatory Authority report. To our knowledge, this study is the most comprehensive one so far applied using WBE for 11 cities in Turkey and evaluates alcohol and nicotine use together.

Wastewater-based surveillance methods have been implemented in several countries as a tool for monitoring SARS-CoV-2 at a community scale. A variety of methods have been used for concentrating, extracting, and detecting the virus, with no clear consensus on the most effective approach. In this note, we report preliminary findings from a study that is tracking SARS-CoV-2 in wastewater in Halifax, Nova Scotia, with a specific focus on the use of four reverse transcriptase quantitative PCR (RT-qPCR) assays for detecting the virus in wastewater. We were able to detect the virus in wastewater samples during the initial rise of cases in the Halifax region in early November 2020. Levels of the targeted SARS-CoV-2 gene fragments increased and fell in response to reported cases of COVID-19. The CDC N1 and E RT-qPCR assays demonstrated greater relative sensitivity than the CDC N2 and N3 assays for detection of SARS-CoV-2 in raw sewage samples.

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.

To assist public health responses to COVID-19, wastewater-based epidemiology (WBE) is being utilised internationally to monitor SARS-CoV-2 infections at the community level. However, questions remain regarding the sensitivity of WBE and its use in low prevalence settings. In this study, we estimated the total number of COVID-19 cases required for detection of SARS-CoV-2 RNA in wastewater. To do this, we leveraged a unique situation where, over a 4-month period, all symptomatic and asymptomatic cases, in a population of approximately 120,000, were precisely known and mainly located in a single managed isolation and quarantine facility (MIQF) building. From 9 July to 6 November 2020, 24-hr composite wastewater samples (n = 113) were collected daily from the sewer outside the MIQF, and from the municipal wastewater treatment plant (WWTP) located 5 km downstream. New daily COVID-19 cases at the MIQF ranged from 0 to 17, and for most of the study period there were no cases outside the MIQF identified. SARS-CoV-2 RNA was detected in 54.0% (61/113) at the WWTP, compared to 95.6% (108/113) at the MIQF. We used logistic regression to estimate the shedding of SARS-CoV-2 RNA into wastewater based on four infectious shedding models. With a total of 5 and 10 COVID-19 infectious cases per 100,000 population (0.005 % and 0.01% prevalence) the predicated probability of SARS-CoV-2 RNA detection at the WWTP was estimated to be 28 and 41%, respectively. When a proportional shedding model was used, this increased to 58% and 87% for 5 and 10 cases, respectively. In other words, when 10 individuals were actively shedding SARS-CoV-2 RNA in a catchment of 100,000 individuals, there was a high likelihood of detecting viral RNA in wastewater. SARS-CoV-2 RNA detections at the WWTP were associated with increasing COVID-19 cases. Our results show that WBE provides a reliable and sensitive platform for detecting infections at the community scale, even when case prevalence is low, and can be of use as an early warning system for community outbreaks., Graphical abstract Image, graphical abstract

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin’s Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin’s Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF’s) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102–1.2 × 109 genome copies/L). Lag analysis of trends in Melvin’s Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15–17 days at the statewide level and by up to 25 days at the regional/county level. Melvin’s Index is a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes and to large regions that are served by multiple WWTFs.

Epidemics require the monitoring of reliable and relevant indicators to inform health care decision making. During the SARS-CoV-2 pandemic, the monitoring of viral dynamics in the population by individual-centered approaches proved to be complex and strongly influenced by asymptomatic carriers and changes in screening policy. In this context, other indicators for global epidemiological monitoring were proposed. Quantification of the virus load in wastewater, using molecular biology tools, quickly emerged as an innovative and objective indicator for monitoring the Covid-19 epidemic. This "wastewater-based epidemiology" (WWBE), whose relevance had been demonstrated a few years earlier, is now a complementary tool for epidemiological monitoring. This indicator is recommended by the European Union since March 2021. The Obépine consortium, which brings together interdisciplinary research teams, has been working on this topic since the beginning of the epidemic in order to develop and master this tool. This research consortium is also the basis of the French national surveillance network of the epidemic via wastewater. The measurement methods, still being optimized in research laboratories, nevertheless require great precautions in their implementation and interpretation for routine use. Through the example of the research program and the network built by Obépine, we will discuss the different steps necessary to set up this epidemiological monitoring via wastewater. The different steps necessary for its mastery are presented here as well as the first lessons to be learned., Les épidémies nécessitent un suivi d’indicateurs fiables et pertinents, afin d’éclairer les prises de décisions sanitaires. Durant la pandémie de SARS-CoV-2, le suivi de la dynamique virale au sein de la population par les approches individu-centrées s’est révélé complexe et fortement influencé par les porteurs asymptomatiques et les modifications de la politique de dépistage. Dans ce contexte, d’autres indicateurs de suivi épidémiologique global ont été très rapidement proposés. La quantification du virus dans les eaux usées, via des outils de biologie moléculaire, s’est très vite révélée comme un indicateur de suivi de l’épidémie de Covid-19 novateur et objectif. Cette « épidé miologie par les eaux usées » (WWBE pour wastewater-based epidemiology en anglais) dont la pertinence avait été démontrée quelques années auparavant, est à présent un outil complémentaire de suivi épidémiologique. Cet indicateur est recommandé par l’Union européenne depuis mars 2021. Le consortium Obépine, regroupant des équipes de recherche interdisciplinaires a, dès le début de l’épidémie, travaillé sur cette thématique afin d’approfondir et de maîtriser cet outil. Ce consortium de recherche est également à la base du réseau de surveillance national français de l’épidémie via les eaux usées. Les méthodes de mesures, encore en cours d’optimisation dans les labo - ratoires de recherche, nécessitent néanmoins de grandes précautions dans leur mise en oeuvre et leur interprétation pour un usage de routine. À travers l’exemple du programme de recherche et du réseau construit par Obépine, nous aborderons ici les différentes étapes nécessaires à la constitution de ce suivi épidémiologique via les eaux usées. Différentes étapes nécessaires à sa maîtrise sont présentées ici ainsi que les premiers enseignements à retenir.

SARS-CoV-2 wastewater-based epidemiology (WBE) has been advancedas a relevant indicator of distribution of COVID-19 in communities, supporting classical testing and tracing epidemiological approaches. An extensive sampling campaign, including ten municipal wastewater treatment plants, has been conducted in different cities of France over a 20-weeks period, encompassing the second peak of COVID-19 outbreak in France. A well-recognised ultrafiltration - RNA extraction - RT-qPCR protocol was used and qualified, showing 5.5 +/-0.5% recovery yield on heat-inactivated SARS-CoV-2. Importantly the whole, solid and liquid, fraction of wastewater was used for virus concentration in this study.Campaign results showed medium- to strong-correlation between SARS-CoV-2 WBE data and COVID-19 prevalence. To go further, WWTP inlet flow rate and raining statistical relationships were studied and taken into account for each WWTP in order to calculate contextualized SARS-CoV-2 loads. This metric presented improved correlation strengths with COVID-19 prevalence for WWTP particularly submitted and sensitive to rain. Such findings highlighted that SARS-CoV-2 WBE data ultimately require to be contextualised for relevant interpretation.HighlightsFirst study monitoring inlet of 10 WWTPs located in France for SARS-CoV-2 RNA quantification over a 20-weeks period encompassing the second peak of COVID-19 outbreakViral recovery yield was 5.5 % +/-0.5% using heat-inactivated SARS-CoV-2Medium to high Spearman’s correlation strength was observed between SARS-CoV-2 WBE and COVID-19 prevalence dataConsidering sampling context (ei. rain events) improved data consistency and correlation strengthGraphical Abstract

Wastewater based epidemiology was employed to track the spread of SARS-CoV-2 within the sewershed areas of 10 wastewater treatment plants (WWTPs) in Catalonia, Spain. A total of 185 WWTPs inflow samples were collected over the period consisting of both the first wave (mid-March to June) and the second wave (July to November). Concentrations of SARS-CoV-2 RNA (N1 and N2 assays) were quantified in these wastewaters as well as those of Human adenoviruses (HAdV) and JC polyomavirus (JCPyV), as indicators of human faecal contamination. SARS-CoV-2 N gene daily loads strongly correlated with the number of cases diagnosed one week after sampling i.e. wastewater levels were a good predictor of cases to be diagnosed in the immediate future. The conditions present at small WWTPs relative to larger WWTPs influence the ability to follow the pandemic. Small WWTPs (, This work was partially funded by the projects PCI2019-103643, RTI2018-097346-B-I00, and AGL2017-86797-C2-1-R projects, all funded by the Spanish Ministry of Science, Innovation and Universities. S. Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona. This study is performed with partial support from the Water Research Institute of the University of Barcelona. IDAEA-CSIC is a Centre of Excellence Severo Ochoa (Spanish Ministry of Science and Innovation, Project CEX2018-000794-S). ICRA authors acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group (ICRA-ENV 2017 SGR 1124). ICRA is part of the CERCA program. We would like to give a big thanks to our families and colleagues, for their support and help behind the scenes.

Down-the-drain disposal of pharmaceuticals remains an overlooked and unrecognized source of environmental contamination that requires nontechnological “at-source” solutions. Monitoring of 31 pharmaceuticals over 7 days in five wastewater treatment plants (WWTPs) serving five cities in South-West UK revealed down-the-drain codisposal of six pharmaceuticals to three WWTPs (carbamazepine and propranolol in city A, sildenafil in city B, and diltiazem, capecitabine, and sertraline in city D), with a one-off record codisposal of estimated 253 pills = 40 g of carbamazepine and estimated 96 pills = 4 g of propranolol in city A accounting for their 10- and 3-fold respective increases in wastewater daily loads. Direct disposal of pharmaceuticals was found to affect the efficiency of wastewater treatment with much higher pharmaceutical removal (decrease in daily load) during “down-the-drain disposal” days. This is due to lack of conjugated glucuronide metabolites that are cleaved during “consumption-only” days, with the release of a parent pharmaceutical counterbalancing its removal. Higher removal of pharmaceuticals during down-the-drain disposal days reduced pharmaceutical loads reaching receiving environment, albeit with significant levels remaining. The estimated daily loads in receiving water downstream from a discharge point accounted for 13.8 ± 3.4 and 2.1 ± 0.2 g day–1 of carbamazepine and propranolol, respectively, during consumption-only days and peaked at 20.9 g day–1 (carbamazepine) and 4.6 g day–1 (propranolol) during down-the-drain disposal days. Actions are needed to reduce down-the-drain disposal of pharmaceuticals. Our recent work indicated that down-the-drain disposal of pharmaceuticals doubled since the last study in 2005, which may be due to the lack of information and messaging that informs people to dispose of unused medicines at pharmacies. Media campaigns that inform the public of how to safely dispose of medicines are key to improving rates of return and reducing pharmaceutical waste in the environment. The environment is a key motivator for returning unused medicines to a pharmacy and so messaging should highlight environmental risks associated with improper disposal., Down-the-drain disposal of pharmaceuticals remains an overlooked and unrecognized source of environmental contamination that requires nontechnological “at-source” solutions.

A thorough understanding of the in-sewer stability of chemical biomarkers is critical in applying wastewater-based surveillance of community drug use. In this study, we examined the effects of sewer biofilm on the degradation of commonly abused drugs, namely, morphine, fentanyl, cocaine, and amphetamine, in wastewater using 48-h batch degradation tests. The experiments were designed to distinguish among abiotic, biochemical, and physical degradation processes, and used mature biofilm obtained from an actual sewer line. Parallel microcosm tests were conducted using wastewater with and without suspended biofilm. Results indicate that first order kinetics describe the degradation of the drugs in both wastewater and wastewater-biofilm microcosms. Amphetamine was most stable in all microcosms, with a maximum removal of only 34% after 48 h. Abiotic chemical transformation played a major role in the degradation of morphine (kab = 0.018 h-1), fentanyl (kab = 0.022 h-1) and cocaine (kab = 0.049 h-1) in wastewater. Fentanyl removal from wastewater was also influenced by the presence of biofilm (kf = 0.015 h-1). This study is the first to report on the effect of sewer biofilm on fentanyl degradation, and highlights the need to account for in-sewer drug stability in wastewater-based drug use estimation, particularly for chemicals with high affinity for organics.

This paper aims to determine whether population normalisation significantly alters the SARS-CoV-2 trends revealed by wastewater-based epidemiology, and whether it is beneficial and/or necessary to provide an understanding of prevalence from wastewater SARS-CoV-2 concentrations. It uses wastewater SARS-CoV-2 data collected from 394 sampling sites, and implements normalisation based on concentrations of a) ammoniacal nitrogen, and b) orthophosphate. Wastewater SARS-CoV-2 metrics are evaluated at a site and aggregated level against three indicators prevalence, based on positivity rates from the Office for National Statistics Coronavirus Infection Survey and test results reported by NHS Test and Trace. Normalisation is shown to have little impact on the overall trends in the wastewater SARS-CoV-2 data on average. However, significant variability between the impact of population normalisation at different sites, which is not evident from previous WBE studies focussed on a single site, is also revealed. Critically, it is demonstrated that while the impact of normalisation on SARS-CoV-2 trends is small on average, it is not reasonable to conclude that it is always insignificant. When averaged across many sites, normalisation strengthens the correlation between wastewater SARS-CoV-2 data and indicators of prevalence; however, confidence in the improvement is low. Lastly, it is noted that most data were collected during periods of national lockdown and/or local restrictions, and thus the impacts and benefits of population normalisation are expected to be higher when normal travel habits resume.

Wastewater-based epidemiology (WBE) has a long history of identifying a variety of viruses from poliovirus to coronaviruses, including novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The presence and detection of SARS-CoV-2 in human feces and its passage into the water bodies are significant public health challenges. Hence, the hot issue of WBE of SARS-CoV-2 in the coronavirus respiratory disease (COVID-19) pandemic is a matter of utmost importance (e.g., SARS-CoV-1). The present review discusses the background, state of the art, actual status, and prospects of WBE, as well as the detection and quantification protocols of SARS-CoV-2 in wastewater. The SARS-CoV-2 detection studies have been performed in different water matrixes such as influent and effluent of wastewater treatment plants, suburban pumping stations, hospital wastewater, and sewer networks around the globe except for Antarctica. The findings revealed that all WBE studies were in accordance with clinical and epidemiological data, which correlates the presence of SARS-CoV-2 ribonucleic acid (RNA) with the number of new daily positive cases officially reported. This last was confirmed via Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR) testing which unfortunately is not suitable for real-time surveillance. In addition, WBE concept may act as a faster protocol to alert the public health authorities to take administrative orders (possible re-emerging infections) due to the impracticality of testing all citizens in a short time with limited diagnostic facilities. A comprehensive and integrated review covering all steps starting from sampling to molecular detection of SARS-CoV-2 in wastewater has been made to guide for the development well-defined and reliable protocols.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater produced interest in its use for sentinel surveillance at a community level and as a complementary approach to syndromic surveillance. With this work, we set the foundations for wastewater-based epidemiology (WBE) in Portugal by monitoring the trends of SARS-CoV-2 RNA circulation in the community, on a nationwide perspective during different epidemiological phases of the pandemic. The Charité assays (E_Sarbecco, RdRP, and N_Sarbecco) were applied to monitor, over 32-weeks (April to December 2020), the dynamics of SARS-CoV-2 RNA at the inlet of five wastewater treatment plants (WWTP), which together serve more than two million people in Portugal. Raw wastewater from three Coronavirus disease 2019 (COVID-19) reference hospitals was also analyzed during this period. In total, more than 600 samples were tested. For the first weeks, detection of SARS-CoV-2 RNA was sporadic, with concentrations varying from 103 to 105 genome copies per liter (GC/L). Prevalence of SARS-CoV-2 RNA increased steeply by the end of May into late June, mainly in Lisboa e Vale do Tejo region (LVT), during the reopening phase. After the summer, with the reopening of schools in mid-September and return to partial face-to-face work, a pronounced increase of SARS-CoV-2 RNA in wastewater was detected. In the LVT area, SARS-CoV-2 RNA load agreed with reported trends in hotspots of infection. Synchrony between trends of SARS-CoV-2 RNA in raw wastewater and daily new COVID-19 cases highlights the value of WBE as a surveillance tool, particularly after the phasing out of the epidemiological curve and when hotspots of disease re-emerge in the population which might be difficult to spot based solely on syndromic surveillance and contact tracing. This is the first study crossing several epidemiological stages highlighting the long-term use of WBE for SARS-CoV-2., Graphical abstract Unlabelled Image

A portion of those infected with SARS-CoV-2 shed the virus and its genetic material in respiratory fluids, saliva, urine, and stool, thus giving the potential to monitor for infections via wastewater. Wastewater surveillance efforts to date have largely assumed that stool shedding has been the primary source of SARS-CoV-2 RNA signal; however, there are increasing questions about the possible contribution of other shedding routes, with implications for wastewater surveillance design and feasibility. In this study we used clinical SARS-CoV-2 RNA shedding data and a Monte Carlo framework to assess the relative contribution of various shedding routes on SARS-CoV-2 RNA loads in wastewater. Stool shedding dominated total SARS-CoV-2 RNA load for community-level surveillance, with mean contributions more than two orders of magnitude greater than other shedding routes. However, RNA loads were more nuanced when considering building-level monitoring efforts designed to identify a single infected individual, where any shedding route could plausibly contribute a detectable signal. The greatest source of model variability was viral load in excreta, suggesting that future modeling efforts may be improved by incorporating specific modeling scenarios with precise SARS-CoV-2 shedding data, and beyond that wastewater surveillance must continue to account for large variability during data analysis and reporting. Importantly, the findings imply that wastewater surveillance at finer spatial scales is not entirely dependent on shedding via feces for sensitive detection of infections thus enlarging the potential use cases of wastewater as a non-intrusive surveillance methodology., Graphical abstract Unlabelled Image

In the current pandemic of COVID-19, sewage surveillance of SARS-CoV-2 genome has been used to complement viral epidemiology in different countries. The aim of this work was to introduce and evaluate this wastewater-based tool in the metropolitan region of Buenos Aires, Argentina. As a pilot study, surveillance of SARS-CoV-2 in wastewater from three districts of this area was performed for more than nine months from June 2020 to April 2021. Viruses present in the samples were concentrated using polyethylene glycol precipitation and quantified using RT-qPCR CDC N1 assay. Virus recovery for SARS-CoV-2 and a potential surrogate, bovine coronavirus Mebus strain, that shares the Betacoronavirus genus and structural characteristics with SARS-CoV-2, were evaluated after concentration and detection procedures. Recovery of both viruses did not differ significantly, with a median for SARS-CoV-2 and BCoV of 0.085 (95% CI: 0.021-0.179) and 0.262 (95% CI: 1.18 × 10-5-0.564) respectively. The concentration of SARS-CoV-2 genome in wastewater ranged from 10 -1 to 10 3 cg/ml, depending on the wastewater treatment plant, type of collection site, viral recovery of the concentration method and the epidemiological situation of the outbreaks. Significant correlations were observed between SARS-CoV-2 concentration in wastewater and reported clinical cases, reinforcing the utility of this approach to monitor the epidemiological status of populations., Graphical abstract Unlabelled Image

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 129 million confirm cases. Many health authorities around the world have implemented wastewater-based epidemiology as a rapid and complementary tool for the COVID-19 surveillance system and more recently for variants of concern emergence tracking. In this study, three SARS-CoV-2 target genes (N1, N2, and E) were quantified from wastewater influent samples (n = 250) obtained from the capital city and 7 other cities in various size in central Ohio from July 2020 to January 2021. To determine human-specific fecal strength in wastewater samples more accurately, two human fecal viruses (PMMoV and crAssphage) were quantified to normalize the SARS-CoV-2 gene concentrations in wastewater. To estimate the trend of new case numbers from SARS-CoV-2 gene levels, different statistical models were built and evaluated. From the longitudinal data, SARS-CoV-2 gene concentrations in wastewater strongly correlated with daily new confirmed COVID-19 cases (average Spearman’s r = 0.70, p < 0.05), with the N2 gene being the best predictor of the trend of confirmed cases. Moreover, average daily case numbers can help reduce the noise and variation from the clinical data. Among the models tested, the quadratic polynomial model performed best in correlating and predicting COVID-19 cases from the wastewater surveillance data, which can be used to track the effectiveness of vaccination in the later stage of the pandemic. Interestingly, neither of the normalization methods using PMMoV or crAssphage significantly enhanced the correlation with new case numbers, nor improved the estimation models. Whole-genome sequencing result showed that those detected SARS-CoV-2 variants of concern from the wastewater matched with the clinical isolates from the communities. The findings from this study suggest that wastewater surveillance is effective in COVID-19 trend tracking and variant emergence and transmission within a community.

Wastewater based epidemiology (WBE) has been successfully applied for SARS-CoV-2 surveillance at the city and building levels. However, sampling at the city level does not provide sufficient spatial granularity to identify COVID-19 hotspots, while data from building-level sampling are too narrow in scope for broader public health application. The objective of this study was to examine the feasibility of using wastewater from wastewater collection systems (WCSs) to monitor COVID-19 hotspots at the zip code level. In this study, 24-h composite wastewater samples were collected from five manholes and two wastewater treatment plants (WWTPs) in the City of Lincoln, Nebraska. By comparing to the reported weekly COVID-19 case numbers, we identified different hotspots responsible for two COVID-19 surges during the study period. One zip code was the only sampling locations that was consistently tested positive during the first COVID-19 surge. In comparison, nearly all the zip codes tested exhibited virus concentration increases that overlapped with the second COVID-19 surge, suggesting broader spread of the virus at that time. These findings demonstrate the feasibility of using WBE to monitor COVID-19 at the zip code level. Highly localized disease surveillance methods can improve public health prevention and mitigation measures at the community level., Graphical abstract Unlabelled Image

Endogenous chemicals specific to human metabolism have been suggested to be good candidates for markers of population size in wastewater-based epidemiology (WBE). So far, creatinine is the only endogenous chemical to be assessed against the criteria of in-sewer stability. This study thus aimed to evaluate the fate of three other endogenous compounds, 5-hydroxy indole acetic acid (5-HIAA), cortisol and androstenedione, under different sewer conditions using laboratory-scale sewer reactors. The results showed that while all compounds were stable in wastewater only (i.e. without biofilm), cortisol and androstenedione degraded quickly in sewers with the presence of sewer biofilms. The degradation followed first-order kinetics similar to that of creatinine. In contrast, 5-HIAA was relatively stable in sewer reactors. This study also recognised the impact of wastewater pH on the detectability of 5-HIAA using a LC-MS/MS direct injection method. In samples acidified to pH 2, the method did not allow routine detection/quantification of 5-HIAA whereas in non-acidified samples the method was sufficiently sensitive for routine quantification of 5-HIAA. The stability of 5-HIAA in sewers and the possibility to measure it using a simple and rapid analytical method corroborate that 5-HIAA may be a suitable biomarker for estimation of population size in WBE.

Wastewater-based epidemiology (WBE) has been consolidated as a tool to provide real-time information on consumption of legal and illegal drugs of abuse by the population. However, WBE is expected to achieve more ambitious objectives such as establishing exposure to certain agents (pesticides, personal care products, persistent organic pollutants, and pathogens), incidence of specific diseases (diabetes, allergies, oxidative stress, and cancer), and determination of some lifestyle consequences (exposure to personal care products, consumption of doping substances or products for the treatment of erectile dysfunction) or environmental factors (increase in temperature) in populations. Herein, we outlined the state-of-the-art applications of WBE including its advantages and disadvantages as well as the prospects for achieving a broader assessment of the habits and health of populations. WBE needs to be further strengthened to help epidemiologists and health authorities to correctly assess fingerprints of human activities in wastewater., This work has been supported by the Generalitat Valenciana (local government of the Valencian Community) through the project ANTROPOCEN@ http://www.antropocena.com (PROMETEO/2018/155).

The determination of biomarkers in sewage samples is a useful tool to monitor the consumption of contaminants of human use evolving to wastewater-based epidemiology (WBE). Solid-phase extraction (SPE) followed by high specific and sensitive determination techniques such as liquid chromatography with mass spectrometry–based detection is usually used for the determination of the low concentration of these biomarkers in such complex samples. The different SPE sorbents and the variables involved in the SPE protocol such as sample loading, washing, and elution solvent are critically reviewed from studies dealing with WBE. Moreover, the results in form of extraction recoveries and matrix effect achieved are discussed. At the end, the review intents to overview the different possibilities of SPE when applied in WBE.

Antibiotics have drawn much attention as their wide usage in humans and animals may result in microbial resistance, which is a huge threat to humans' health. Studies on the occurrence and removals of antibiotics in wastewater treatment plants have been widely performed, but very few covered their main acetylated metabolites. This study developed an effective analytical method for the trace determination of four sulfonamides and three acetylated metabolites in municipal wastewaters, which was validated by linearity (R2 > 0.995), sensitivity (limit of quantification, LOQ

The correction factor (CF) is a critical parameter for back-estimating the consumption of a drug via wastewater-based epidemiology (WBE). The CF is usually the excretion factor (EF) of the drug or ...

Ketamine (KET) analogs are increasingly emerging as new psychoactive substances (NPS). The present report describes the first detection of the KET analog, 2-fluorodeschloroketamine (2F-DCK), in influent samples collected from nine wastewater treatment plants in seven major Chinese cities from 2018 to 2020 by wastewater-based epidemiology (WBE). An analytical method based on solid-phase extraction and subsequent gas chromatography-mass spectrometry was developed for the detection of 2F-DCK and KET. The stability experiments showed that 2F-DCK and KET remained stable in wastewater for 15 days at room and frozen temperatures, and at two pH values (pH = 7 and pH = 2), with residue amounts between 90% and 110%. KET was detected in all samples, whereas 2F-DCK was detected in only four samples: from Guangzhou in 2018, Shenzhen in 2019, and Quanzhou and Nanning in 2020, indicating that 2F-DCK has been used as early as 2018 in China. The renal clearance of 2F-DCK was predicted based on the quantitative structure-pharmacokinetic relationship model, which was used to calculate an excretion factor of 3.7. The 2F-DCK consumption in four cities ranged from 3.71 ± 0.05 to 55 ± 0.09 mg/day/1000 inh, and KET ranged from 1.3 ± 0.04 to 76.5 ± 4.63 mg/day/1000 inh. This is the first study to investigate 2F-DCK by WBE, which provides relevant real-time data on the growth of NPS use, as well as useful information for the government to develop new policies.

New methodologies based on the principle of “sewage epidemiology” have been successfully applied before in the detection of illegal drugs. The study describes the idea of early detection of a virus, e.g., SARS-CoV-2, in wastewater in order to focus on the area of virus occurrence and supplement the results obtained from clinical examination. By monitoring temporal variation in viral loads in wastewater in combination with other analysis, a virus outbreak can be detected and its spread can be suppressed early. The use of biosensors for virus detection also seems to be an interesting application. Biosensors are highly sensitive, selective, and portable and offer a way for fast analysis. This manuscript provides an overview of the current situation in the area of wastewater analysis, including genetic sequencing regarding viral detection and the technological solution of an early warning system for wastewater monitoring based on biosensors.

A national wastewater-based epidemiology (WBE) monitoring campaign has been initiated to determine the trends in drug consumption in Turkey since 2019. This study aimed to present the wastewater monitoring results for four periods in 2019 for six major cities with 17 wastewater treatment plants. The study investigated heroin (HER), amphetamine (AMP), methamphetamine (METH), cocaine (COC), 3,4-methylenedioxymethamphetamine (MDMA), cannabis, alcohol, and tobacco to understand the temporal and geographical drug consumption trends. The results indicated a trend of regular cannabis use in all the cities. Considering geographical variations, the drug usage trends were not homogeneous within the country for any of the investigated drugs, except cannabis. HER consumption was predominant in Denizli (1042.48 mg/1000 inh/day), while Izmir and Antalya (154.10 and 79.56 mg/1000 inh/day, respectively) topped for COC consumption. Aydin had the highest consumption levels of AMP (90.03 mg/1000 inh/day) and METH (358.13 mg/1000 inh/day), while MDMA consumption was the highest in Samsun (157.64 mg/1000 inh/day). Tobacco consumption was high in Aydin (8791.69 mg/1000 inh/day) and Antalya (5375.17 mg/1000 inh/day), whereas alcohol consumption was higher in Bursa than in the other cities (53434.61 mL/1000 inh/day). There were no statistically significant differences in the weekend consumption levels of the investigated drugs among these six cities. Although this study included the results of 1 year of consumption monitoring, the data confirms internationally published information on the drug trafficking routes of conventional drugs in this region, especially the Heroin-Balkan route. Results from further sampling will enable accurate evaluation of global drug consumption and trafficking, in addition to alcohol and tobacco use.

Wastewater-based epidemiology (WBE) was utilized to monitor SARS-CoV-2 RNA in sewage collected from manholes specific to individual student dormitories (dorms) at the University of Arizona in the fall semester of 2020, which led to successful identification and reduction of SARS-CoV-2 transmission events. Positive wastewater samples triggered clinical testing of residents within that dorm; thus, SARS-CoV-2 infected individuals were identified regardless of symptom expression. This current study examined clinical testing data to determine the abundance of asymptomatic versus symptomatic cases in these defined communities. Nasal and nasopharyngeal swab samples processed via antigen and PCR tests indicated that 79.2% of SARS-CoV-2 infections were asymptomatic, and only 20.8% of positive cases reported COVID-19 symptoms at the time of testing. Clinical data was paired with corresponding wastewater virus concentrations, which enabled calculation of viral shedding rates in feces per infected person. Mean shedding rates averaged from positive wastewater samples across all dorms were 7.30 ± 0.67 log10 genome copies per gram of feces (gc/g-feces) based on the N1 gene. Quantification of SARS-CoV-2 fecal shedding rates from infected individuals has been the critical missing component necessary for WBE models to measure and predict SARS-CoV-2 infection prevalence in communities. The findings from this study can be utilized to create models that can be used to inform public health prevention and response actions., Graphical abstract Unlabelled Image

Clinical testing of children in schools is challenging, with economic implications limiting its frequent use as a monitoring tool of the risks assumed by children and staff during the COVID-19 pandemic. Here, a wastewater based epidemiology approach has been used to monitor 16 schools (10 primary, 5 secondary and 1 post-16 and further education for a total of 17 sites) in England. A total of 296 samples over 9 weeks have been analysed for N1 and E genes using qPCR methods. Of the samples returned, 47.3% were positive for one or both genes with a frequency of detection in line with the respective community. WBE offers a promising low cost, non-invasive approach for supplementing clinical testing and can offer longitudinal insights that are impractical with traditional clinical testing.

Wastewater-based epidemiology (WBE) is expected to become a powerful tool to monitor the dissemination of SARS-CoV-2 at the community level, which has attracted the attention of scholars all over the world. However, there is not yet a standard protocol to guide its implementation. In this paper, we proposed a comprehensive technical and theoretical framework of relative quantification via qPCR for determining the virus abundance in wastewater and estimating the infection ratio in corresponding communities, which is expected to achieve horizontal and vertical comparability of the data using a human-specific biomarker as the internal reference. Critical factors affecting the virus detectability and the estimation of infection ratio include virus concentration methods, lag-period, per capita virus shedding amount, sewage generation rate, temperature-related decay kinetics of virus/biomarker in wastewater, and hydraulic retention time (HRT), etc. Theoretical simulation shows that the main factors affecting the detectability of virus in sewage are per capita virus shedding amount and sewage generation rate. While the decay of SARS-CoV-2 RNA in sewage is a relatively slow process, which may have limited impact on its detection. Under the ideal condition of high per capita virus shedding amount and low sewage generation rate, it is expected to detect a single infected person within 400,000 people., Graphical abstract Unlabelled Image

Wastewater is a complex matrix containing a wide range of chemical and biological markers of human activity. Relating concentrations of these “waste” materials in wastewater influent streams to population-scale use, consumption, or rates of exposure, can provide important qualitative and/or quantitative information on the combined activity of inhabitants within a given wastewater catchment. This approach has become known as wastewater-based epidemiology (WBE) and is widely applied for monitoring community use of selected pharmaceuticals, illicit drugs, tobacco and alcohol. However, many other potential applications are emerging that can contribute useful knowledge on human health, exposure to industrial chemicals, infectious diseases or pathogens, antibiotic resistance and other catchment-based activities.This presentation will discuss the role pf sensor-based approaches for the field’s future development.

Graphical abstract, Wastewater-based epidemiology (WBE) is a promising approach for estimating population-wide COVID-19 prevalence through detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. However, various methodological challenges associated with WBE would affect the accuracy of prevalence estimation. To date, the overall uncertainty of WBE and the impact of each step on the prevalence estimation are largely unknown. This study divided the WBE approach into five steps (i.e., virus shedding; in-sewer transportation; sampling and storage; analysis of SARS-CoV-2 RNA concentration in wastewater; back-estimation) and further summarized and quantified the uncertainties associated with each step through a systematic review. Although the shedding of SARS-CoV-2 RNA varied greatly between COVID-19 positive patients, with more than 10 infected persons in the catchment area, the uncertainty caused by the excretion rate became limited for the prevalence estimation. Using a high-frequency flow-proportional sampling and estimating the prevalence through actual water usage data significantly reduced the overall uncertainties to around 20–40% (relative standard deviation, RSD). And under such a scenario, the analytical uncertainty of SARS-CoV-2 RNA in wastewater was the dominant factor. This highlights the importance of using surrogate viruses as internal or external standards during the wastewater analysis, and the need for further improvement on analytical approaches to minimize the analytical uncertainty. This study supports the application of WBE as a complementary surveillance strategy for monitoring COVID-19 prevalence and provides methodological improvements and suggestions to enhance the reliability for future studies.