Wastewater-based epidemiology can be used to analyse substances in wastewater and serve as a tool to examine the illegal drug consumption in a population. The aim of this study was to analyse concentrations of illegal drugs in wastewater from Öns wastewater treatment plant in Umeå, Sweden and i) compare concentrations of illegal drugs with previous studies from 11 and 9 years ago, ii) examine the weekly variation and iii) examine how Umeå's levels of drugs in wastewater relate to other cities in Sweden and Europe. The method used is flow proportional sampling. Three samples were collected on Monday (24-hours), Wednesday (24-hours) and a combined sample from the weekend (Friday, Saturday, Sunday, i.e., 72-hours), the samples were transferred to vials containing sodium thiosulfate (Na2S2O3), they were transported refrigerated to a laboratory in Prague, Czech Republic, where the analysis was performed. This study gives strong indications that cocaine use has increased in Umeå over the past decade and is thus in line with the increasing trend of cocaine being measured in wastewater in other European cities. This study also provides indications that the use of amphetamine has increased in Umeå over the last decade and supports previously observed trends of high amphetamine levels in Sweden in comparison with the rest of Europe. The analysis indicates higher cocaine use on weekends compared to weekdays, while amphetamine use was more evenly distributed over the week.

A rural-urban divide in health status and healthcare infrastructure has been well-documented in the U.S., where populations residing in census regions classified as rural often exhibit more negative health outcomes, adverse health behaviors, and have reduced access to affordable and proximal health services, compared to their urban and peri-urban counterparts. However, it is important to note that such disparities vary based on specific rural regions and individual circumstances. Rural areas may face elevated risk factors for infectious diseases such as increased proximity to wildlife and livestock and disproportionately high reliance on private, non-federally regulated, primary drinking water sources. Chronic conditions prevalent in rural communities such as diabetes and hypertension are frequently linked with longer duration and higher severity of symptoms than in urban areas; this association suggests that the risk of exposure to infectious diseases and the likelihood of progression to serious illness and hospitalization may be elevated, although this is not universally the case across all rural settings. Alongside documented urban-rural health disparities, there also exist disparities in the nature and quality of data on health-related behaviors, outcomes, and service provision in rural areas compared to urban and peri-urban regions. In this dissertation, two key environmental matrices –drinking water and wastewater– were highlighted as vectors of information to better estimate levels of contaminant exposures and health outcomes in rural communities. First, baseline data on drinking water contaminant levels and associated health outcome data were highlighted as crucial for refining holistic exposure estimates as well as understanding drinking water related health burdens in rural communities where a larger proportion of households use private drinking water sources, such as well water, that are not federally regulated. Second, systematic sampling and testing of pathogen biomarkers in wastewater to non-invasively measure population-level health status, also known as wastewater based surveillance (WBS) and, depending on the context, wastewater based epidemiology (WBE) is not constrained by disadvantages of clinical testing, e.g., limited health-care access, long travel times to testing facilities, delay between symptom-onset and testing. Thus, expanded implementation of WBS in rural communities is proposed here as a strategy to address data disparities in clinical testing for infectious diseases. Collectively, this dissertation advances knowledge on estimated drinking water contaminant levels, exposures, and associated public health outcomes and corresponding research gaps in rural Appalachian U.S., and elucidates pathways toward best practices and considerations for public-health focused wastewater testing adoption in rural communities. For the latter, the question of whether WBS challenges unique to rural wastewater systems hinder application of WBS in small, rural communities was explored, as well as methods to advance best-practices for rural WBS. To summarize existing publicly available peer-reviewed literature on drinking water contaminants in rural Appalachian U.S., in Chapter 2, a systematic review and meta-analysis of microbial and chemical drinking water contaminants was performed. Key contaminants were identified as being elevated beyond regulatory, health-based, maximum contaminant levels in our meta-analyses from rural drinking water sources in Appalachia, including E coli, lead, arsenic, uranium. Overall, we found data on drinking water source quality under baseline conditions (i.e., rather than post anomalous contamination events such as chemical spills) in rural Appalachian U.S. was sparse relative to widespread media coverage on the issue. Epidemiologic-based research studies that collected both drinking water exposure data and paired health outcome data were also limited. As a result, although some instances of anomalously high levels of drinking water contaminants were identified in rural Appalachia from the published literature, we could not obtain a clear picture of baseline exposures to drinking water contaminants in most rural Appalachian communities, highlight need to address these knowledge gaps. In Chapter 3, to evaluate whether wastewater could serve as a reliable metric for estimating community circulation of viruses and antimicrobial resistance (AMR) markers, even when sourced from aging and low-resource sewer collection networks, a 12-month wastewater monitoring study was conducted in a small, rural sewer conveyance system with pronounced infrastructural challenges. Specifically, the field site under study was compromised with heavy inflow and infiltration (IandI). Detection rates and concentrations of viral, AMR, and human fecal markers were grouped by levels of IandI impact across the sewershed, and location-, date-, and sample- specific variables were assessed for their relative influence on viral, AMR, and human fecal marker signal using generalized linear models (GLMs). We found that while IandI likely adversely impacted the magnitude of wastewater biomarker signal to some extent throughout the sewershed, especially up-sewer at sites with more pronounced IandI, substantial diminishment of wastewater signal at WWTP influent was not observed in response to precipitation events. Thus, our data indicated that WWTP influent sampling alone can still be used to assess and track community circulation of pathogens in heavily IandI impacted systems, particularly for ubiquitously circulating viruses less prone to dilution induced decay. Delineations were also made for what circumstances up-sewer sampling may be necessary to better inform population shedding of pathogens, especially where IandI is prevalent. Various normalization strategies have been proposed to account for sources of variability for deriving population-level pathogen shedding from wastewater, including those introduced by IandI-driven dilution. Thus, in Chapter 4, we evaluated the temporal and spatial variability of viral and AMR marker signal in wastewater at different levels of IandI, both unnormalized and with the adoption of several normalization strategies. We found that normalization using physicochemical-based wastewater strength metrics (chemical oxygen demand, total suspended solids, phosphate, and ammonia) resulted in higher temporal and site-specific variability of SARS-CoV-2 and human fecal biomarker signal compared to unnormalized data, especially for viral and AMR marker signal measured in wastewater from sites with pronounced IandI. Viral wastewater signal normalized to physicochemical wastewater strength metrics and flow data also closely mirrored precipitation trends, suggesting such normalization approaches may more closely scale wastewater trends towards precipitation patterns rather than per capita signal in an IandI compromised system. We also found that in most cases, normalization did not significantly alter the relationship between wastewater trends and clinical infection trends. These findings suggest a degree of caution is warranted for some normalization approaches, especially where precipitation driven IandI is heightened. However, data and findings largely supported the utility of using human fecal markers such as crAssphage for normalizing wastewater signal to address site-specific differences in dilution levels, since viral signal scaled to this metric did not result in strong correlations between precipitation and wastewater trends, higher spatial and temporal variation was not observed, and strong correlations were observed between viral signal and viral infection trends. Finally, in chapter 5, we assessed the relationship between monthly Norovirus GII, Rotavirus, and SARS-CoV-2 wastewater trends with seasonal infection trends for each of the viruses to ascertain whether WBE could be used in a rural sewershed of this size with substantial IandI impacts to track and potentially predict population level infection trends. Though up-sewer, or near-source sampling, at sites with permanent IandI impacts did not exhibit a clear relationship with seasonal infection trends for Rotavirus, SARS-CoV-2, and Norovirus GII, WWTP influent signal and consensus signals aggregated from multiple up-sewer sites largely mirrored expected seasonal trends. Findings also suggested that for more ubiquitous viral targets, such as SARS-CoV-2, viral trends measured at WWTP influent in a small IandI impacted system may still provide a sufficiently useful measure of infection trends to inform the use of WBE (assuming appropriate normalization to sewershed population). These findings elucidate the potential utility and relative robustness of wastewater testing to ascertain community-level circulation of pathogens in small, rural sewersheds even those compromised by extensive IandI inputs. Overall, this dissertation examined drinking water and wastewater as critical metrics for assessing contaminant exposures and infectious disease trends in rural communities, particularly in the context of small, rural communities which tend to have more limited health infrastructure and lower-resource wastewater systems. Overall, findings underscore the need for baseline data on drinking water quality by identifying gaps in current knowledge and calling for further research to better understand drinking water contaminant exposure levels in rural areas. Wastewater as a non-invasive, population-level health metric was evaluated in the context of a small, rural sewer system overall, and by varying observed levels of IandI, as well as associated tradeoffs for normalization adoption. By evaluating these environmental surveillance metrics using both desk-based and field-based research study designs, findings from this dissertation offer valuable insights and practical recommendations for improving baseline drinking water quality monitoring and wastewater pathogen testing, all with the overarching goal of supporting more targeted public health interventions in rural settings. / Doctor of Philosophy / In the United States, there is a significant health and healthcare gap between rural and urban areas. Rural communities often face worse health outcomes, poorer health behaviors, and have less access to affordable and nearby healthcare services compared to their urban and peri-urban counterparts. Additionally, rural areas are exposed to higher risks for infectious diseases due to closer proximity to wildlife and livestock and proportionately lower access to regulated drinking water sources. Chronic conditions like diabetes and hypertension, which are more common in rural populations, can exacerbate the severity and duration of symptoms for infectious diseases, potentially leading to more serious illness and hospitalizations. Despite these heightened risks, data on health behaviors, outcomes, and healthcare services in rural areas is often lacking and less comprehensive compared to urban regions. This dissertation investigates two promising avenues of improving monitoring to provide information needed to better understand and address contaminant exposures and health trends in rural communities: drinking water and wastewater. Firstly, this dissertation underscores the importance of establishing baseline data on drinking water quality. This is essential for accurately estimating exposure levels and understanding the health impacts associated with elevated levels of drinking water contaminants, particularly in rural areas where a higher share of primary drinking water sources is unregulated by the federal government compared to urban areas. This study reveals significant gaps in current knowledge and highlights the need for more research to provide a clearer picture of drinking water quality in these communities. Secondly, this dissertation explores the use of wastewater as a non-invasive tool for assessing community health. This method, known as wastewater-based surveillance (WBS) or wastewater-based epidemiology (WBE), offers a way to measure population-level health trends without relying on clinical testing, which can be limited by factors such as access to healthcare and delays in testing. The dissertation evaluates how effective wastewater monitoring can be in small, rural sewer systems, even when these systems face challenges like aging infrastructure and significant inflow and infiltration (IandI) from groundwater and surface water. It examines how different normalization strategies for wastewater data can influence the reliability of this method and how wastewater testing can be adapted to account for varying levels of IandI. Overall, the dissertation provides valuable insights into the effectiveness of using drinking water and wastewater as environmental metrics for informing public health intervention strategies in rural settings. It offers justifications for improving drinking water quality monitoring and wastewater testing practices, aiming to support more targeted and effective public health interventions in rural communities. By addressing the challenges and limitations associated with these environmental monitoring strategies this research contributes to a better understanding of how to reduce health data disparities in rural areas.

Following the detection of SARS-CoV-2 RNA fragments in wastewater in March 2020, the use of wastewater-based epidemiology is rapidly emerging as a non-invasive approach to assessing community wide COVID-19 prevalence. Wastewater-based epidemiology is promoted as a complement to clinical testing in terms of improving public health authorities’ preparedness and supporting them in taking required interventions to protect the public health.  To utilize the occurrence of SARS-CoV-2 in wastewater in a public health context and evaluate trends in viral prevalence on a community level, robust data interpretation is required. This master's thesis work examines what is currently known about the factors that influence the concentrations of SARS-CoV-2 in wastewater, as well as the impact of alternative normalization approaches on the relationship between viral wastewater data and clinical case numbers. The work is divided into two parts where this report provides a state-of-the-art review as well as an overview of key findings in the accompanied scientific paper.  The study involved collection of wastewater samples from two different sized wastewater treatment plants (WWTPs) in Luleå municipality, between January and March 2021.  Concentrations of SARS-CoV-2 RNA were quantified in each sample as well as concentrations of total nitrogen (TN), total phosphorus (TP) and pepper mild mottle virus (PMMoV). Average daily WWTP flow and environmental data (precipitation and temperature) were continuously measured over the sampling period. The population size was estimated based on mass loads of TN and TP and literature values of the domestic contribution of these parameters to the sewer system. The viral wastewater data was normalized using the estimations of population size, census data, WWTP flow and PMMoV. The impact of the alternative normalization approaches was evaluated using statistical analyses to explore the relationship to clinical case numbers.  The result showed that the strength of the correlation between the different normalization approaches and clinical case data differed between the WWTPs. However, within each WWTP the differences in correlation between the different normalization approaches and clinical cases were not significant. Moreover, normalization using WWTP flow (i.e., viral loads) showed a stronger correlation to clinical cases compared to population and PMMoV normalized viral loads. However, when comparing wastewater data between the two catchment areas, the normalization approaches utilizing population (either census or TN and TP estimated population) or PMMoV better reflect the number of clinical cases. Additionally, according to time-shifted analysis, wastewater data of SARS-CoV-2 RNA predated an increase in clinical cases by 0-2 and 5-8 days, respectively, for the larger and smaller WWTPs.

During the past three years, we assisted to the rise of a new pathogen that afflicted the world with a global pandemic. Working in an era of rapid change has posed important challenges and the focus of research has shifted more and more toward topics of greater social utility. However, this period has also brought a new role for wastewater highlighting how it can provide insight into the health of a community. This is the approach of Wastewater-based Epidemiology (WBE). The work presented here aimed to deepen this approach not only at the theoretical level but also contributing with an ongoing monitoring of about 30 months. The main objectives were (i) to collect information on the recently discovered SARS-CoV-2 virus, its biology, transmission mechanism, and role in wastewater treatment plants (WWTP); (ii) set up a surveillance system that would allow to monitor SARS-CoV-2 infections over time, obtaining early information on its spread among the population to support the Health Authority. Starting from a detailed study of the shedding mechanisms of SARS-CoV-2 in the feces of infected patients, we moved on to the evaluation of the viral concentrations in the sewage system and the wastewater entering the WWTP. The possibility of a faecal-oral transmission route of the virus was investigated by evaluating the data about viability and infectivity in wastewater. The natural processes of decay of the virus in wastewater and the reduction of its concentration in the different treatment stages of WWTPs were explored in literature and with experimental data. At the same time, we developed a SARS-CoV-2 surveillance system in wastewater by applying different detection methods. Some practical and scientific aspects of the analysis protocol have been studied in depth such as the choice of the type of sample, the storage temperatures, and the pre-heat treatments aimed at making the analysis safer for the operator. The choice of the concentration method was evaluated to comply with the low concentration of the viral titer and therefore the crucial importance of this phase of the protocol. During the monitoring campaign, we further investigated aspects related to data processing and developed normalization approaches. Samples from WWTPs in the province of Trento were analysed weekly and sampling frequencies and curve smoothing methods deriving from those data were evaluated. The trend curves thus obtained were compared with those deriving from clinical data provided by the local Health Authority and signals of early warnings of virus diffusion trends in the population were highlighted. With the alternation of the different variants of the virus and the evidence of their importance in the development of new waves of infection, a PCR based genotyping method has been devised to rapidly identify the already known variants. In conclusion, this research project addressed a broad spectrum of aspects related to the WBE approach in contrasting the COVID-19 emergency and confirmed that wastewater could be a valuable source of information and management support for this and other emerging pathogens or micropollutants.

abstract: This dissertation critically evaluated methodologies and devices for assessing and protecting the health of human populations, with particular emphasis on groundwater remediation and the use of wastewater-based epidemiology (WBE) to inform population health. A meta-analysis and assessment of laboratory-scale treatability studies for removing chlorinated solvents from groundwater found that sediment microcosms operated as continuous-flow columns are preferable to batch bottles when seeking to emulate with high fidelity the complex conditions prevailing in the subsurface in contaminated aquifers (Chapter 2). Compared to monitoring at the field-scale, use of column microcosms also showed (i) improved chemical speciation, and (ii) qualitative predictability of field parameters (Chapter 3). Monitoring of glucocorticoid hormones in wastewater of a university campus showed (i) elevated stress levels particularly at the start of the semester, (ii) on weekdays relative to weekend days (p = 0.05) (161 ± 42 μg d-1 per person, 122 ± 54 μg d-1 per person; p ≤ 0.05), and (iii) a positive association between levels of stress hormones and nicotine (rs: 0.49) and caffeine (0.63) consumption in this student population (Chapter 4). Also, (i) alcohol consumption determined by WBE was in line with literature estimates for this young sub-population (11.3 ± 7.5 g d-1 per person vs. 10.1 ± 0.8 g d-1 per person), whereas caffeine and nicotine uses were below (114 ± 49 g d-1 per person, 178 ± 19 g d-1 per person; 627 ± 219 g d-1 per person, 927 ± 243 g d-1 per person). The introduction of a novel continuous in situ sampler to WBE brought noted benefits relative to traditional time-integrated sampling, including (i) a higher sample coverage (93% vs. 3%), (ii) an ability to captured short-term analyte pulses (e.g., heroin, fentanyl, norbuprenorphine, and methadone), and (iii) an overall higher mass capture for drugs of abuse like morphine, fentanyl, methamphetamine, amphetamine, and the opioid antagonist metabolite norbuprenorphine (p ≤ 0.01). Methods and devices developed in this work are poised to find applications in the remediation sector and in human health assessments. / Dissertation/Thesis / Doctoral Dissertation Civil, Environmental and Sustainable Engineering 2018

Under covid-19-pandemin har avloppsvattenbaserad epidemiologi (WBE) använts i stor utsträckning som ett komplement till kliniska tester över många delar av världen. Detta projekt syftade till att detektera och kvantifiera belastningen av Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) i avloppsvattenprover med hjälp av Revers transkriptas kvantitativ polymeraskedjereaktion (RT-qPCR). De analyserade proverna kom från fyra olika avloppsreningsverk i Sverige, under perioden november 2022 till maj 2023. Studien omfattade en översikt över olika provtagnings- och analytiska tekniker och normaliseringsmetoder som används i WBE-studier, vilket betonade vikten av metodval. SARS-CoV-2-RNA upptäcktes i alla analyserade prover och infektionstrender kunde identifieras effektivt, inklusive COVID-19-vågen som observerades under semesterperioden. De dominerande varianterna som upptäcktes under denna övervakningsperiod var omikron variantens undergrupper, BA.2. och BA.2.75. Den veckovisa kvantifierade SARS-CoV-2-belastningen i avloppsvattenproverna visade en signifikant positiv korrelation till de kliniska fall som rapporterats i motsvarande avrinningsområden. Denna associering förstärktes ytterligare genom att normalisera SARS-CoV-2-innehållet med fekal biomarkör peppar milt fläckvirus (PMMoV). Dessutom har två metoder för tidig varning, nämligen medelvärdet plus två standardavvikelser (MSD) och positiv procentuell förändring (PPC), implementerats på avloppsvattendata, vilket pekar på vikten av att tillämpa sådana varningsmetoder för att ge förståeliga och tolkbara resultat. Denna studie ger värdefulla insikter om övervakning och analys av SARS-CoV-2 i avloppsvatten, vilket bidrar till utvecklingen av robusta system för tidig varning och folkhälsostrategier. / During the COVID-19 pandemic, wastewater-based epidemiology (WBE) has been applied extensively as a complementary tool to clinical testing across many parts of the globe. This project aimed to detect and measure the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) load in wastewater samples using Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The analyzed samples were from four different wastewater treatment plants (WWTPs) in Sweden, covering the period from November 2022 through May 2023. The study encompassed an overview of various sampling and analytical techniques and normalization approaches employed in WBE studies, highlighting the importance of method selection. SARS-CoV-2 RNA was detected in all the samples analyzed, and infection trends could be identified effectively, including the COVID-19 peak observed during the holiday season. The dominant variants detected during this monitoring period were the omicron variants; omicron BA.2. and omicron BA.2.75. The weekly quantified SARS-CoV-2 load in the wastewater samples showed a significant positive correlation to the clinical cases reported in the corresponding catchment areas. This association was further enhanced by normalizing SARS-CoV-2 content with the fecal biomarker pepper mild mottle virus (PMMoV). Furthermore, two early warning methods, namely the mean plus two standard deviations (MSD) and positive percentage change (PPC), were implemented on the wastewater data pinpointing the importance of applying such warning methods to provide understandable and interpretable results. This study provides valuable insights into the monitoring and analysis of SARS-CoV-2 in wastewater, contributing to the development of robust early warning systems and public health strategies.

Tesis por compendio / [ES] El objetivo inicial de esta tesis era detectar y rastrear virus entéricos en diferentes matrices acuáticas mediante la combinación de protocolos moleculares y metagenómicos. Se establecieron como objetivos principales el desarrollo de procedimientos para la concentración de virus en muestras de aguas residuales, el análisis de virus indicadores de contaminación fecal y la caracterización del viroma de estas muestras. En cuanto al desarrollo de procedimientos para la concentración de virus entéricos emergentes en muestras de aguas residuales, el protocolo de floculación con hidróxido de aluminio, utilizado habitualmente en el grupo para la concentración de otros virus entéricos, resultó muy eficaz para el virus de la Hepatitis E, permitiendo su detección en muestras de aguas de entrada y salida de estaciones depuradoras de aguas residuales (EDAR). Por lo que respecta al seguimiento de la prevalencia de otros virus entéricos e indicadores virales en muestras de entrada y salida de EDARs, esta tesis proporciona información cuantitativa sobre la presencia del indicador crAssphage y otros virus entéricos de cápside intacta en aguas de diferentes EDARs valencianas. Además, los resultados de la correlación indican que crAssphage podría no ser un indicador óptimo de la presencia de virus entéricos infecciosos en las aguas residuales tratadas. En relación a la caracterización del viroma de las muestras de agua de las EDARs analizadas, en la presente tesis se describe un procedimiento de referencia que permite la detección y caracterización de las poblaciones virales en las muestras de aguas residuales recogidas a la entrada y salida de la planta depuradora. También se refleja el sesgo existente en los perfiles del viroma que se obtienen según las librerías de secuenciación que se empleen. En este sentido, esta investigación arroja luz sobre la diversidad de las comunidades virales en influentes y efluentes de aguas residuales, proporcionando información valiosa también en términos de indicadores fecales virales. Con la llegada de la pandemia de COVID-19 a principios de 2020, el SARS-CoV-pasó a ser el protagonista de la segunda parte de la tesis. En este aspecto, se marcaron como objetivos implementar un sistema de monitorización de SARS-CoV-2 en aguas residuales y desarrollar y optimizar métodos moleculares rápidos para inferir la infectividad del SARS-CoV-2. Los resultados obtenidos han demostrado que la aplicación de la epidemiología basada en aguas residuales (WBE) es eficiente para estimar la presencia de COVID-19 en comunidades y puede servir de herramienta para la salud pública como alerta temprana ante situaciones pandémicas. Asimismo, esta tesis incluye el primer estudio publicado en España que realizó un análisis metagenómico de la diversidad del SARS-CoV-2 presente en las aguas residuales en las tres primeras oleadas epidemiológicas, cuyos resultados confirmaron el potencial de la secuenciación masiva de aguas residuales para detectar nuevas mutaciones y linajes del SARS-CoV-2. Además, también se han comparado y optimizado los protocolos de concentración, extracción y detección de ácidos nucleicos de coronavirus a partir de muestras de aguas residuales, superficiales y de mar. Así, este trabajo amplía el conocimiento sobre los procedimientos analíticos para la detección del SARS-CoV-2 en aguas residuales favoreciendo la implementación global del COVID-19 WBE. Finalmente, se ha implementado un protocolo de RT-qPCR de viabilidad basado en el cloruro de platino para evitar la amplificación del ARN del SARS-CoV-2 no infeccioso. Además, los resultados de esta tesis apoyan la idea de que el SARS-CoV-2 presente en las aguas residuales no es infeccioso. En general, en el marco de esta tesis doctoral se ha desarrollado una herramienta analítica rápida basada en la RT-qPCR de viabilidad para inferir la infectividad del SARS-CoV-2 con potencial aplicación en la evaluación de riesgos, la prevención y el control en los programas de salud / [CA] L'objectiu inicial d'esta tesi era detectar i rastrejar virus entèrics en diferents matrius aquàtiques mitjançant la combinació de protocols moleculars i metagenòmics. Es van establir com a objectius específics el desenvolupament de procediments per a la concentració de virus en mostres d'aigües residuals, l'anàlisi de virus indicadors de contaminació fecal i la caracterització del viroma d'estes mostres. Pel que fa al desenvolupament de procediments per a la concentració de virus entèrics emergents en mostres d'aigües residuals, el protocol de floculació amb hidròxid d'alumini, utilitzat habitualment al grup per a la concentració d'altres virus entèrics, va resultar molt eficaç per al virus de l'hepatitis E, permetent-ne la detecció en mostres d'aigües d'entrada i de sortida d'estacions depuradores d'aigües residuals (EDAR). Pel que fa al seguiment de la prevalença d'altres virus entèrics i indicadors virals en mostres d'entrada i de sortida d'EDARs, esta tesi proporciona informació quantitativa sobre la presència de l'indicador crAssphage i altres virus entèrics de càpside intacta en aigües de diferents EDARs valencianes. A més, els resultats de la correlació indiquen que el bacteriòfag crAssphage podria no ser un indicador òptim de la presència de virus entèrics infecciosos a les aigües residuals tractades. Pel que fa a la caracterització del viroma de les mostres d'aigua de les EDARs analitzades, a la present tesi es descriu un procediment de referència que permet la detecció i caracterització de les poblacions virals a les mostres d'aigües residuals recollides a l'entrada i sortida de la planta depuradora. També es reflecteix el biaix existent als perfils del viroma que s'obtenen segons les llibreries de seqüenciació que s'utilitzen. En este sentit, esta investigació aporta coneixement sobre la diversitat de les comunitats virals en influents i efluents d'aigües residuals, proporcionant informació valuosa també en termes d'indicadors fecals virals. Amb l'arribada de la pandèmia de COVID-19 a principis del 2020, el SARS-CoV-va passar a ser el protagonista de la segona part de la tesi. En este aspecte, es van establir com a objectius implementar un sistema de monitorització de SARS-CoV-2 en aigües residuals i desenvolupar i optimitzar mètodes moleculars ràpids per inferir la infectivitat del SARS-CoV-2. Els resultats obtinguts han demostrat que l'aplicació de l'epidemiologia basada en aigües residuals (WBE) és eficient per estimar la presència de COVID-19 a les comunitats i pot servir d'eina per a la salut pública com a alerta primerenca davant de situacions pandèmiques. Així mateix, esta tesi inclou el primer estudi publicat a Espanya que va realitzar una anàlisi metagenòmica de la diversitat del SARS-CoV-2 present a les aigües residuals a les tres primeres onades epidemiològiques, els resultats del qual van confirmar el potencial de la seqüenciació massiva d'aigües residuals per a detectar noves mutacions i llinatges del SARS-CoV-2. A més, també s'han comparat i optimitzat els protocols de concentració, extracció i detecció d'àcids nucleics de coronavirus a partir de mostres d'aigües residuals, superficials i de mar. Així, este treball amplia el coneixement sobre els procediments analítics per a la detecció del SARS-CoV-2 en aigües residuals afavorint la implementació global del COVID-19 WBE. Finalment, s'ha implementat un protocol de RT-qPCR de viabilitat basat en el clorur de platí per evitar l'amplificació de l'ARN del SARS-CoV-2 no infecciós. A més, els resultats d'esta tesi donen suport a la idea que el SARS-CoV-2 present a les aigües residuals no és infecciós. En general, en el marc d'esta tesi doctoral s'ha desenvolupat una eina analítica ràpida basada en la RT-qPCR de viabilitat per inferir la infectivitat del SARS-CoV-2 amb potencial aplicació en l'avaluació de riscos, la prevenció i el control en els programes de Salut Pública. / [EN] The initial aim of this thesis was to detect and monitor the presence of enteric viruses in different aquatic matrices using both molecular and metagenomic protocols. The specific objectives were the development of procedures for the concentration of viruses in wastewater samples, the analysis of viruses indicative of faecal contamination and the characterisation of the virome in these samples. Regarding the development of procedures for the concentration of emerging enteric viruses in wastewater samples, the flocculation protocol with aluminium hydroxide, commonly used in the group for the concentration of other enteric viruses, proved to be very effective for Hepatitis E virus, allowing its detection in influent and effluent water samples from wastewater treatment plants (WWTPs). Regarding the monitoring of the prevalence of other enteric viruses and viral indicators in incoming and outgoing samples from WWTPs, this thesis provides quantitative information on the presence of the indicator crAssphage and other enteric viruses with intact capsid in water from different Valencian WWTPs. Moreover, the correlation results indicate that crAssphage may not be an optimal indicator of the presence of infectious enteric viruses in treated wastewater. In relation to the virome characterisation of the WWTP water samples analysed, the present thesis describes a reference procedure that allows the detection and characterisation of viral populations in wastewater. It also reflects the existing bias in the virome profiles obtained depending on the sequencing libraries used. In this sense, this research sheds light on the diversity of viral communities in influent and effluent wastewater, providing valuable information also in terms of faecal viral indicators. With the advent of the COVID-19 pandemic in the early 2020s, SARS-CoV-2 became the focus of the second part of the thesis. In this aspect, the objectives were to implement a monitoring system for SARS-CoV-2 in wastewater and to develop and optimise rapid molecular methods to infer SARS-CoV-2 infectivity. The results obtained have demonstrated that the application of wastewater-based epidemiology (WBE) is efficient for estimating the presence of COVID-19 in communities and can serve as a Public Health tool for early warning of pandemic situations. Furthermore, this thesis includes the first study published in Spain that performed a metagenomic analysis of the SARS-CoV-2 diversity present in wastewater in the first three epidemiological waves, the results of which confirmed the potential of mass sequencing of wastewater to detect new SARS-CoV-2 mutations and lineages. In addition, protocols for the concentration, extraction and detection of coronavirus nucleic acids from sewage, surface and seawater samples have also been compared and optimised. Thus, this work expands the knowledge on analytical procedures for the detection of SARS-CoV-2 in wastewater favouring the global implementation of the COVID-19 WBE. Finally, a viability RT-qPCR protocol based on platinum chloride has been implemented to avoid amplification of non-infectious SARS-CoV-2 RNA. Furthermore, the results of this thesis support the idea that SARS-CoV-2 present in wastewater is not infectious. Overall, in the framework of this PhD thesis, a rapid analytical tool based on feasibility RT-qPCR has been developed to infer the infectivity of SARS-CoV-2 with potential application in risk assessment, prevention and control in Public Health programmes. / This thesis has been funded by an "Ayudas para contratos predoctorales para la formación de doctores 2018” grant, awarded by the Ministry of Science and Innovation. / Cuevas Ferrando, E. (2022). Detection and tracking of emerging viruses of public health interest in waters through molecular and metagenomic procedures [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/190899 / Compendio

Fecal shedding of SARS-CoV-2 leads to a renaissance of wastewater-based epidemiology (WBE) as additional tool to follow epidemiological trends in the catchment of treatment plants. As alternative to the most commonly used composite samples in surveillance programs, passive sampling is increasingly studied. However, the many sorbent materials in different reports hamper the comparison of results and a standardization of the approach is necessary. Here, we compared different cost-effective sorption materials (cheesecloths, gauze swabs, electronegative filters, glass wool, and tampons) in torpedo-style housings with composite samples. Despite a remarkable variability of the concentration of SARS-CoV-2-specific gene copies, analysis of parallel-deposited passive samplers in the sewer demonstrated highest rate of positive samples and highest number of copies by using cheesecloths. Using this sorption material, monitoring of wastewater of three small catchments in the City of Dresden resulted in a rate of positive samples of 50% in comparison with composite samples (98%). During the investigation period, incidence of reported cases of SARS-CoV-2 in the catchments ranged between 16 and 170 per 100,000 persons and showed no correlation with the measured concentrations of E gene in wastewater. In contrast, constantly higher numbers of gene copies in passive vs. composite samples were found for human adenovirus and crAssphage indicating strong differences of efficacy of methods concerning the species investigated. Influenza virus A and B were sporadically detected allowing no comparison of results. The study contributes to the further understanding of possibilities and limits of passive sampling approaches in WBE.

Dependent on the excretion pattern, wastewater monitoring of viruses can be a valuable approach to characterizing their circulation in the human population. Using polyethylene glycol precipitation and reverse transcription-quantitative PCR, the occurrence of RNA of SARS-CoV-2 and influenza viruses A/B in the raw wastewater of two treatment plants in Germany between January and May 2022 was investigated. Due to the relatively high incidence in both exposal areas (plant 1 and plant 2), SARS-CoV-2-specific RNA was determined in all 273 composite samples analyzed (concentration of E gene: 1.3 × 10⁴ to 3.2 × 10⁶ gc/L). Despite a nation-wide low number of confirmed infections, influenza virus A was demonstrated in 5.2% (concentration: 9.8 × 10² to 8.4 × 10⁴ gc/L; plant 1) and in 41.6% (3.6 × 10³ to 3.0 × 10⁵ gc/L; plant 2) of samples. Influenza virus B was detected in 36.0% (7.2 × 10² to 8.5 × 10⁶ gc/L; plant 1) and 57.7% (9.6 × 10³ to 2.1 × 10⁷ gc/L; plant 2) of wastewater samples. The results of the study demonstrate the frequent detection of two primary respiratory viruses in wastewater and offer the possibility to track the epidemiology of influenza by wastewater-based monitoring.

Globalt har hela världens befolkning påverkats både ekonomiskt och psykiskt av coronaviruset SARS-CoV-2, som har drabbat så många människor med covid-19 att det klassas som en pandemi. Strax efter pandemins utbrott upptäcktes det att viruset utsöndras från avföring och ut i spillvattennätet som leder till reningsverken. Då virusmängden ökar i avloppsvattnet ökar även covid-19 fallen i samhället. Ökning av virusmängd i avloppsvatten kan nämligen signalera om att det förekommer smittspridning i samhället. Avloppsvattenbaserad övervakning kan dock användas som komplement till andra teststrategier vilket EU-kommissionen har nämnt i en rekommendation. Trender kan analyseras för att i ett tidigt skede informera sjukvård och regioner om ökad smittspridning.  På Kungsängens reningsverk i Västerås kommun har Mälarenergi analyserat avloppsvattnet för att ta reda på om ökning av virus i avloppsvatten kan indikera på ökad smittspridning i Västerås kommun. De har samlat in proverna och skickat det till SGS Analytics AB Sweden som har analyserat proverna med RT-qPCR. CT-värdena har normaliserats med vattenflöden. Korrelationstest har gjorts mellan virusmängd i avloppsvattnet och covid-19 fall, dödsfall samt IVA-fall. Det fanns ett signifikant svagt negativt samband mellan virusmängd i avloppsvatten och covid-19 fall per vecka. Mellan virusmängd och IVA-fall eller dödsfall fanns inget samband. Det finns en del felkällor som kan ha påverkat virusmängden. Vid höga vattenflöden kan PCR inhibitorer från tillskottsvatten och lakvatten ha påverkat CT-värdena. Värdena är höga på sommaren trots att covid-19 fall, dödsfall och IVA-fall var som lägst. Inhibitorer skapar direkt eller indirekt högre CT-värden vilket tolkas som lägre virusmängder.