Wastewater-based Epidemiology Research Articles

Longitudinal wastewater-based surveillance of SARS-CoV-2 during 2023 in Ethiopia

IntroductionAlthough wastewater-based epidemiology (WBE) successfully functioned as a tool for monitoring the coronavirus disease 2019 (COVID-19) pandemic globally, relatively little is known about its utility in low-income countries. This study aimed to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater, estimate the number of infected individuals in the catchment areas, and correlate the results with the clinically reported COVID-19 cases in Addis Ababa, Ethiopia.MethodsA total of 323 influent and 33 effluent wastewater samples were collected from three Wastewater Treatment Plants (WWTPs) using a 24-h composite Moore swab sampling method from February to November 2023. The virus was captured using Ceres Nanotrap® Enhancement Reagent 2 and Nanotrap® Microbiome A Particles, and then nucleic acids were extracted using the Qiagen QIAamp Viral RNA Mini Kit. The ThermoFisher TaqPath™ COVID-19 kit was applied to perform real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to quantify the SARS-CoV-2 RNA. Wastewater viral concentrations were normalized using flow rate and number of people served. In the sampling period, spearman correlation was used to compare the SARS-CoV-2 target gene concentration to the reported COVID-19 cases. The numbers of infected individuals under each treatment plant were calculated considering the target genes’ concentration, the flow rate of treatment plants, a gram of feces per person-day, and RNA copies per gram of feces.ResultsSARS-CoV-2 was detected in 94% of untreated wastewater samples. All effluent wastewater samples (n = 22) from the upflow anaerobic sludge blanket (UASB) reactor and membrane bioreactor (MBR) technology were SARS-COV-2 RNA negative. In contrast, two out of 11 effluents from Waste Stabilization Pond were found positive. Positive correlations were observed between the weekly average SARS-CoV-2 concentration and the cumulative weekly reported COVID-19 cases in Addis Ababa. The estimated number of infected people in the Kality Treatment catchment area was 330 times the number of COVID-19 cases reported during the study period in Addis Ababa.DiscussionThis study revealed that SARS-CoV-2 was circulating in the community and confirmed previous reports of more asymptomatic COVID-19 cases in Ethiopia. Additionally, this study provides further evidence of the importance of wastewater-based surveillance in general to monitor infectious diseases in low-income settings.ConclusionWastewater-based surveillance of SARS-CoV-2 can be a useful method for tracking the increment of COVID-19 cases before it spreads widely throughout the community.

A-233 Wastewater-based surveillance for SARS-CoV-2 Subvariants using the Nanopore Sequencing in South Korea

Abstract Background Genomic tools to identify and characterize SARS-CoV-2 and its various subvariants were adapted from clinical settings. Wastewater-based surveillance (WBS) has since been recognized as tool of transformative potential, providing real-time objective, comprehensive data on the prevalence and distribution of the target pathogen across the space and time. We conducted wastewater-based epidemiology (WBE) at sewage treatment plants to find a more efficient mutation monitoring method, and respond to pre-existing infectious diseases. Methods In our study, domestic sewage collected every weeks from 6 sewage treatment plants in urban region from June 2023 to November 2023 was used to concentrate and isolate viruses using wastewater ultra nucleic acid isolation kits with virus enrichment kits. Positive samples confirmed were subjected to SARS-CoV-2 whole genome sequencing (WGS) using GridION (Oxford Nanopore Technologies). The experimental data was aligned through Epi2me, analyzed using Nextclade, that was compared with KCDC official reports and our monitoring results (total 780 cases) ongoing at SCL. Results Various mutations were identified at each collection time, occurring new subvariants over time. The mutations (orf1a:A690V, S:F456L) sequentially confirmed from the sample collected on Aug. 23rd are specific mutations of XBB.1.9.2 and EG.5. In addition, the mutations orf1b: D54N, S:L455F, which were sequentially confirmed from the sample collected on Sep. 6th, are HK.3, a descendent lineage of EG.5, and FLip variants, which show growth advantage, were confirmed. Furthermore, the detection rate of FLip variants detected from Sep.6th gradually increased from 17% to 67% (Fig.1). Taken together, we confirmed the similarity between monthly detected mutations and changes in dominant species over time by comparing with ‘monthly COVID-19 mutation trends’ and ‘HK.3 dominance announcement by KCDC’(Fig.2). Conclusions WBS would be a useful tool for proactively responding to infectious diseases including COVID-19 by exploring new mutations and predicting changes in dominant species.

Tracking epidemic viruses in wastewaters.

Classical epidemiology relies on incidence, mortality rates, and clinical data from individual testing, which can be challenging for many countries. Therefore, innovative, flexible, cost-effective, and scalable surveillance techniques are needed. Wastewater-based epidemiology (WBE) has emerged as a highly powerful tool in this regard. WBE analyses substances excreted in human fluids and faeces that enter the sewer system. This approach provides insights into community health status and lifestyle habits. WBE serves as an early warning system for viral surveillance, detecting the emergence of new pathogens, changes in incidence rates, identifying future trends, studying outbreaks, and informing the performance of action plans. While WBE has long been used to study different viruses such as poliovirus and norovirus, its implementation has surged due to the pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2. This has led to the establishment of wastewater surveillance programmes at international, national, and community levels, many of which remain operational. Furthermore, WBE is increasingly applied to study other pathogens, including antibiotic resistance bacteria, parasites, fungi, and emerging viruses, with new methodologies being developed. Consequently, the primary focus now is on creating international frameworks to enhance states' preparedness against future health risks. However, there remains considerable work to be done, particularly in integrating the principles of One Health into epidemiological surveillance to acknowledge the interconnectedness of humans, animals, and the environment in pathogen transmission. Thus, a broader approach to analysing the three pillars of One Health must be developed, transitioning from WBE to wastewater and environmental surveillance, and establishing this approach as a routine practice in public health.

Evaluating survey techniques in wastewater-based epidemiology for accurate COVID-19 incidence estimation

Wastewater-based epidemiology (WBE) requires high-quality survey methods to determine the incidence of infections in wastewater catchment areas. In this study, the wastewater survey methods necessary for comprehending the incidence of infection by WBE are clarified. This clarification is based on the correlation with the number of confirmed coronavirus disease 2019 (COVID-19) cases, considering factors such as handling non-detect data, calculation method for representative values, analytical sensitivity, analytical reproducibility, sampling frequency, and survey duration. Data collected from 15 samples per week for two and a half years using a highly accurate analysis method were regarded as gold standard data, and the correlation between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater and confirmed COVID-19 cases was analyzed by Monte Carlo simulation under the hypothetical situation where the quality of the wastewater survey method was reduced. Regarding data handling, it was appropriate to replace non-detect data with estimates based on distribution, and to use geometric means to calculate representative values. For the analysis of SARS-CoV-2 RNA in samples, using a highly sensitive and reproducible method (non-detect rates of <40 %; ≤0.4 standard deviation) and surveying at least three samples, preferably five samples, per week were considered desirable. Furthermore, conducting the survey over a period of time that included at least 50 weeks was necessary. A WBE that meets these survey criteria is sufficient for the determination of the COVID-19 infection incidence in the catchment. Furthermore, WBE can offer additional insights into infection rates in the catchment, such as the estimated 48 % decrease in confirmed COVID-19 cases visiting a clinic following a COVID-19 legal reclassification in Japan.

Wastewater surveillance in post-omicron silent phase uncovers silent waves and cryptic transmission of SARS-CoV-2 variants; a yearlong study in Western India

Due to reduced clinical testing and evolving monitoring challenges, tracking the emergence and evolution of SARS-CoV-2 variants has become increasingly complex. To address this gap, we investigated the utility of wastewater-based epidemiology (WBE) as a complementary tool for SARS-CoV-2 variant surveillance in sewage treatment plants (STPs) across Pune, India. We analyzed 1128 wastewater samples collected between May 2022 and May 2023, using Illumina and nanopore sequencing techniques for robust detection and variant characterization. The study revealed critical findings, including “silent waves” with elevated viral load despite minimal clinical cases, suggesting potential cryptic transmission. These silent waves aligned with the dominance of Omicron BA.2 in June–July 2022 and emergence of the recombinant XBB clade in December 2022. Importantly, sequencing detected XBB lineages 130–253 days before their initial clinical identification, demonstrating its significant advantage in early variant detection. Furthermore, wastewater analysis revealed a higher degree of lineage diversity compared to clinical data, indicating its ability to capture a broader spectrum of circulating variants. The BA.2.86.X was identified 103 days prior to its clinical detection in Pune, highlighting WBE's remarkable lead time. Surprisingly, BF.7.X and BQ.X fragments were also detected in wastewater but not yet reported clinically. These findings demonstrate the remarkable value of WBE as an early warning tool for SARS-CoV-2 variants ahead of time. By revealing silent waves, enabling early variant detection, and capturing a broader viral spectrum, WBE effort could empower public health officials to make informed decisions and implement effective strategies to mitigate future waves, especially in contexts with declining clinical testing.

Wastewater monitoring of human and avian influenza A viruses in Northern Ireland: a genomic surveillance study

Influenza Aviruses (IAVs) are significant pathogens of humans and other animals. Although endemic in humans and birds, novel IAV strains can emerge, jump species, and cause epidemics, like the latest variant of H5N1. Wastewater-based epidemiology (WBE) has been shown capable of detecting human IAVs. We aimed to assess whether whole-genome sequencing (WGS) of IAVs from wastewater is possible and can be used to discriminate between circulating strains of human and any non-human IAVs, such as those of avian origin. Using a pan-IAV RT-quantitative PCR assay, six wastewater treatment works (WWTWs) across Northern Ireland were screened from Aug 1to Dec 5, 2022. Ananopore WGS approach was used to sequence RT-qPCR-positive samples. Phylogenetic analysis of sequences relative to currently circulating human and non-human IAVs was performed. Forcomparative purposes, clinical data (PCR test results) were supplied by The Regional Virus Laboratory, Belfast Health and Social Care Trust (Belfast, Northern Ireland, UK). We detected a dynamic IAV signal in wastewater from Sept 5, 2022, onwards across Northern Ireland, which did not show a clear positive relationship with the clinical data obtained for the region. Meta (mixed strain) whole-genome sequences were generated from wastewater samples displaying homology to only human and avian IAVstrains. The relative proportion of IAV reads of human versus avian origin differed across time and sample site. Adiversity in subtypes and lineages was detected (eg, H1N1, H3N2, and several avian). Avian segment 8related to those found in recent H5N1 clade 2.3.4.4b was identified. WBE affords a means to monitor circulating human and avian IAV strains and provide crucial genetic information. As such, WBE can provide rapid, cost-effective, year-round One Health surveillance to help control IAV epidemic and pandemic-related threats. However, optimisation of WBE protocols are necessary to ensure observed wastewater signals not only correlate with clinical case data, but yield information on the wider environmental pan-influenz-ome. Department of Health for Northern Ireland.

Early Introductions of Candida auris Detected by Wastewater Surveillance, Utah, USA, 2022-2023.

Candida auris is considered a nosocomial pathogen of high concern and is currently spreading across the United States. Infection control measures for C. auris focus mainly on healthcare facilities, yet transmission levels may already be significant in the community before outbreaks are detected in healthcare settings. Wastewater-based epidemiology (culture, quantitative PCR, and whole-genome sequencing) can potentially gauge pathogen transmission in the general population and lead to early detection of C. auris before it is detected in clinical cases. To learn more about the sensitivity and limitations of wastewater-based surveillance, we used wastewater-based methods to detect C. auris in a southern Utah jurisdiction with no known clinical cases before and after the documented transfer of colonized patients from bordering Nevada. Our study illustrates the potential of wastewater-based surveillance for being sufficiently sensitive to detect C. auris transmission during the early stages of introduction into a community.

Persistence of respiratory, enteric, and fecal indicator viruses in fecal sludge from on-site sanitation in Dakar, Senegal

ABSTRACT As wastewater-based epidemiology (WBE) broadens to include prevalent diseases with significant global health impact, existing surveillance systems concentrate on sewer-based infrastructure, which excludes the 2.7 billion people using non-sewered systems. To address this gap, our study explores the potential of fecal sludge treatment plants for WBE, emphasizing the stability of virus RNA targets within pooled fecal sludge. We screened fecal sludge from a centralized treatment facility in Dakar, Senegal for SARS-CoV-2, human norovirus, and microbial source trackers pepper mild mottle virus and tomato brown rugose fruit virus (ToBRFV). Decay kinetics of viral genomic RNA markers were examined at 4, 15, and 30 °C over 70 days. Results indicate the high persistence of viral targets in fecal sludge (T90 value of 3.3 months for exogenous SARS-CoV-2 N1 and N2 and 6.2 months for ToBRFV), with all targets detected throughout the 70-day experiment under various temperatures with limited decay (&amp;lt;90% reduction). This study addresses a crucial gap in understanding virus persistence in on-site sanitation systems by providing essential decay rate constants for effective target detection. Our results indicate that sampling at centralized facilities treating fecal sludge from on-site sanitation could facilitate localized pathogen surveillance in low-income settings.

Multi-biomarker approach for estimating population size in national-scale wastewater-based epidemiology studies

This study identifies biochemical markers (BCIs) that can be used as population markers in wastewater-based epidemiology (WBE) and compares their estimates with other established population size estimation (PE) methods, including census data (PECEN). Several groups of BCIs (64 targets: genetic and chemical markers) were investigated in an intercity study, including 10 cities/towns within England equating to a population of ∼7 million people. Several selection criteria were applied to identify the best BCIs to provide robust estimation of population size at a catchment level: (1) excellent performance with analytical methods; (2) excellent fit of the linear regression model which indicates PE-driven BCI daily loads; (3) low temporal variability in usage; (4) human-linked origin. Only a few tested BCIs showed a strong positive linear correlation between daily BCI loads and PE indicating their low spatiotemporal variability. These are: cimetidine, clarithromycin, metformin, cotinine, bezafibrate, metronidazole and hydroxymetronidazole, diclofenac, and benzophenone 1. However, only high/long term usage pharmaceuticals: cimetidine and metformin as well as cotinine (metabolite of nicotine) performed well when tested in two independent datasets and catchments accounting for both spatial and temporal scales. Strong seasonal usage trends were observed for antihistamines, NSAIDs (anti-inflammatories), antibiotics and UV filters, invalidating them as PE markers. Key conclusions from the study are: (1) Cimetidine is the best performing BCI; (2) Chemical markers outperform genetic markers as PE BCIs; (3) Water utility PE estimates (PEWW) align well with PECEN and PEBCI values; (4) Ammonium/orthophosphate as well as viral PE markers suffer from high temporal variability, hence, they are not recommended as PEBCI markers, and, most importantly, (5) PEBCI calibration/validation at the country/region level is advised in order to establish the best PE markers suited for local/national needs and accounting for site/region specific uncertainties.

Integrated environmental and clinical surveillance for the prevention of acute respiratory infections (ARIs) in indoor environments and vulnerable communities (Stell-ARI): Protocol.

The epidemiological relevance of viral acute respiratory infections (ARIs) has been dramatically highlighted by COVID-19. However, other viruses cannot be neglected, such as influenza virus, respiratory syncytial virus, human adenovirus. These viruses thrive in closed spaces, influenced by human and environmental factors. High-risk closed communities are the most vulnerable settings, where the real extent of viral ARIs is often difficult to evaluate, due to the natural disease progression and case identification complexities. During the COVID-19 pandemic, wastewater-based epidemiology has demonstrated its great potential for monitoring the circulation and evolution of the virus in the environment. The "Prevention of ARIs in indoor environments and vulnerable communities" study (Stell-ARI) addresses the urgent need for integrated surveillance and early detection of ARIs within enclosed and vulnerable communities such as long-term care facilities, prisons and primary schools. The rapid transmission of ARIs in such environments underscores the importance of comprehensive surveillance strategies to minimise the risk of outbreaks and safeguard community health, enabling proactive prevention and control strategies to protect the health of vulnerable populations. This study consists of designing and validating tools for integrated clinical and environmental-based surveillance for each setting, coupled with analytical methods for environmental matrices. The clinical surveillance involves specialized questionnaires and nasopharyngeal swabs for virus identification, while the environmental surveillance includes air and surface microbiological and chemical monitoring, and virological analysis of wastewater. Integrating this information and the collection of behavioural and environmental risk factors into predictive and risk assessment models will provide a useful tool for early warning, risk assessment and informed decision-making. The study aims to integrate clinical, behavioural, and environmental data to establish and validate a predictive model and risk assessment tool for the early warning and risk management of viral ARIs in closed and vulnerable communities prior to the onset of an outbreak.

Wastewater-based epidemiology for COVID-19 surveillance and beyond: A survey

The pandemic of COVID-19 has imposed tremendous pressure on public health systems and social economic ecosystems over the past years. To alleviate its social impact, it is important to proactively track the prevalence of COVID-19 within communities. The traditional way to estimate the disease prevalence is to estimate from reported clinical test data or surveys. However, the coverage of clinical tests is often limited and the tests can be labor-intensive, requires reliable and timely results, and consistent diagnostic and reporting criteria. Recent studies revealed that patients who are diagnosed with COVID-19 often undergo fecal shedding of SARS-CoV-2 virus into wastewater, which makes wastewater-based epidemiology for COVID-19 surveillance a promising approach to complement traditional clinical testing. In this paper, we survey the existing literature regarding wastewater-based epidemiology for COVID-19 surveillance and summarize the current advances in the area. Specifically, we have covered the key aspects of wastewater sampling, sample testing, and presented a comprehensive and organized summary of wastewater data analytical methods. Finally, we provide the open challenges on current wastewater-based COVID-19 surveillance studies, aiming to encourage new ideas to advance the development of effective wastewater-based surveillance systems for general infectious diseases.

Wastewater-Based Epidemiology of SARS-CoV-2 RNA in Bethlehem, PA and Lehigh University

Wastewater-Based Epidemiology of SARS-CoV-2 RNA in Bethlehem, PA and Lehigh University

The first detection of SARS-CoV-2 RNA in the wastewater of Bucharest, Romania

Wastewater-based epidemiology (WBE) has been previously used as a tool for pathogen identification within communities. After the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) outbreak, in 2020, Daughton proposed the implementation of a wastewater surveillance strategy that could determine the incidence of COVID-19 (coronavirus disease 2019) nationally. Individuals in various stages of SARS-CoV-2 infection, including presymptomatic, asymptomatic and symptomatic patients, can be identified as carriers of the virus in their urine, saliva, stool and other bodily secretions. Studies using this method were conducted to monitor the prevalence of the virus in high-density populations, such as cities but also in smaller communities, such as schools and college campuses. The aim of this pilot study was to assess the feasibility and effectiveness of wastewater surveillance in Bucharest, Romania, and wastewater samples were collected weekly from seven locations between July and September 2023. RNA (ribonucleic acid) extraction, followed by dPCR (digital polymerase chain reaction) analysis, was performed to detect viral genetic material. Additionally, NGS (next generation sequencing) technology was used to identify the circulating variants within the wastewater of Bucharest, Romania. Preliminary results indicate the successful detection of SARS-CoV-2 RNA in wastewater, providing valuable insights into the circulation of the virus within the community.

A key factor in monitoring cannabis consumption trends through wastewater analysis: Partitioning of THC[sbnd]COOH between the liquid and solid phase of influent wastewater

Current wastewater-based epidemiology (WBE) studies are predominantly focused on the analysis of urinary biomarkers present in the liquid phase of influent wastewater (IWW). This approach systematically underestimates less polar metabolites, such as cannabis biomarkers. These biomarkers can potentially sorb to and desorb from suspended particulate matter (SPM) present in IWW. This study investigates the bidirectional partitioning of THCCOOH between the liquid phase and SPM of IWW by performing multiple sorption experiments using THCCOOH-D9 as a surrogate due to the unavailability of blank SPM and blank IWW. In addition, this study involves the analysis of IWW collected from eight wastewater treatment plants (WWTP) (n = 56) across four European countries, where raw IWW, the liquid phase and SPM were separately analysed to identify potential trends in the fraction of THCCOOH in the SPM between and withing the examined locations. Based on the performed sorption experiments, bidirectional partitioning of THCCOOH between the liquid phase and SPM was noted, showing partition between both phases when only one of the two phases was spiked. It was illustrated that the concentration of SPM had a notable influence on the THCCOOH partitioning between both phases. In addition to the inter location variability (average THCCOOH present in the SPM ranged 31–59 %), a substantial intra location variation was also observed, e.g., in one location ranged 17–58 %. While the determination of a correction factor for the amount of THCCOOH present in SPM would be ideal, this is challenging, since the amount of SPM is not fixed in all IWW samples. Although SPM has influence on the THCCOOH partition, no correlation (p value Spearman correlation = 0.3160) was observed between the SPM concentration and the fraction of THCCOOH in the solid phase. Moreover, the collection of homogenized samples is difficult, and the time required to reach an equilibration in partitioning of THCCOOH between both IWW phases remains unclear. Due to i) the large inter-and intra-location variation of THCCOOH present in the SPM, ii) the variability in SPM concentration in IWW samples, and iii) the time required to reach a partitioning equilibration, an analytical procedure based on liquid-liquid extraction (LLE) that considers both the liquid phase and SPM of IWW is recommended to reduce the overall uncertainty for THCCOOH measurement in IWW. It was illustrated that this extraction method is capable to recover the total concentration of THCCOOH in both phases.

Wastewater-based evaluation of the efficacy of oxycodone regulations in Australia.

Between 2018 and 2020, Australia implemented major policy changes to improve the quality and safety of opioid prescribing, with a specific focus on oxycodone. This study used wastewater-based epidemiology to assess the efficacy of Australia's regulatory reforms by measuring change in consumption of oxycodone via exploratory analysis. Wastewater analysis data on oxycodone consumption was from the National Wastewater Drug Monitoring Program. The program captures data from more than 50 wastewater treatment plant catchments across Australia, equivalent to more than 50% of the national population. Geographic trend analyses were conducted for both major cities and regional areas within all states and territories of Australia over a 6-year period between 2017 and 2023. Oxycodone consumption showed a statistically significant increase nationally from 78 mg/day/1000 people (95% confidence interval [CI] = 71, 84) in 2017 to 120 mg/day/1000 people in August 2019 (95% CI = 110, 120), an increase of 52% (95% CI = 42, 62, P < 0.0001). From August 2019 to December 2020, there was a statistically significant decrease from 120 to 65 mg/day/1000 people (95% CI = 60, 71), a decrease of 45% (95% CI = 40, 51), followed by a modest 2.4% increase to the end of the study period in April 2023 (95% CI [2.0,2.7]). A 45% reduction in oxycodone consumption in Australia from 2019 to 2020 coincided with national policy changes that aimed to reduce consumption of prescription opioids. The overall declining trend in consumption was suggestive of the effectiveness of national interventions in reducing pharmaceutical opioid use. Wastewater-based epidemiology provides an effective approach for assessing the effectiveness of controlled substances policy changes.

Wastewater-based epidemiology to assess pharmaceutical consumption. Spanish perspective

Wastewater-based epidemiology (WBE) is a well-established approach that can provide objective and real-time data on the consumption of substances such as pharmaceuticals. However, most of the studies reported so far compares consumption data obtained using WBE with those derived from prescription data from public health systems, which is often incomplete and might represent a source of uncertainty.This study aims to compare the measured pharmaceutical consumption back calculated with the WBE approach with consumption derived from dispensed pharmaceuticals in two regions of Spain, managed by two different Health Systems. To do so, a group of 17 pharmaceuticals, including the most representative ones of every therapeutic family, were monitored in influent wastewater (IWW) samples collected over a week campaign in spring 2022 at four different wastewater treatment plants (WWTPs) in Spain: two WWTPs in Madrid city (center of Spain) and two WWTPs in Catalonia (Northeast of Spain).Population-normalized daily loads (PNDL) revealed that the patterns of pharmaceutical occurrence in the different WWTPs are very similar, being acetaminophen, 4-acetamidoantipyrine and valsartan the pharmaceuticals with the highest PNDL values: 17162 ± 1457 mg day−1 1000 inh−1 for acetaminophen, 2365 ± 696 and 2429 ± 263 mg day−1 1000 inh−1 for 4-acetamidoantipyrine, 2006 ± 541 and 2041 ± 352 mg day−1 1000 inh−1 for valsartan.Pharmaceutical PNLDs were then transformed into measured pharmaceutical consumption (MC) and compared with dispensed consumption (DC) data obtained from the pharmacies in the catchment area where the WWTPs are located. A ratio MC/DC within 0.8 to 1.2 was obtained for 11 out of the 17 studied pharmaceuticals. Highlighting a match in all the cardiovascular system pharmaceuticals, with the exception of losartan (1.29–1.39 ratio) and valsartan (1.35–1.43) in all WWTPs. In summary, the degree of correlation between MC/DC is higher than those previously reported comparing with the prescribed pharmaceutical consumption.

Harnessing non-standard nucleic acids for highly sensitive icosaplex (20-plex) detection of microbial threats.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. We benchmarked this assay using a low-cost, portable sequencing platform (Oxford Nanopore) on wastewater, soil, and human stool samples. Using parallelized multi-target TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay, there was 74% agreement on positive calls and 97% agreement on negative calls. Additionally, we show how sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish sub-species. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

International Workers' Day: Consumption Patterns of Morphine, Codeine, and Methamphetamine in Urban and Rural Areas Based on Wastewater-Based Epidemiology.

Wastewater-based epidemiology (WBE) is a reliable means to estimate drug consumption in a specific population. By measuring the concentration of drug residues or metabolites in wastewater, the consumption behavior pattern of a specific population can be deduced. Using the WBE method, the present study, for the first time, continuously monitored the differences in the consumption of morphine (MOR), codeine (CODE), and methamphetamine (METH) in three wastewater-treatment plants in a city and two surrounding villages in Xinjiang, China during International Workers' Day and the following week. The wastewater samples were pretreated by solid-phase extraction and then analyzed by high-performance liquid chromatography-tandem mass spectrometry. Methamphetamine was not detected in rural areas and was detected only on International Workers' Day in urban areas. According to the estimation of per capita consumption, the per capita consumption of MOR, CODE, and METH in urban inhabitants was 12.04 to 23.39, 10.44 to 16.39, and 1.31 mg/day/1000 inhabitants. The per capita consumption of MOR and CODE in rural areas was 5.19 to 8.35 and 2.56 to 3.52 mg/day/1000 inhabitants. The consumption of MOR in urban and rural areas was significantly higher than that of CODE and METH. During International Workers' Day, workdays, and weekends, the consumption of MOR and CODE in urban areas is significantly higher than that in rural areas. Compared with those on weekends, the consumption of urban MOR and CODE increased more during International Workers' Day. The consumption of MOR in urban areas showed a weekend effect. The present study can provide information for subsequent research and government departments. Environ Toxicol Chem 2024;00:1-9. © 2024 SETAC.

Assessing different next-generation sequencing technologies for wastewater-based epidemiology

Wastewater-based epidemiology has proven to be an important public health asset during the COVID-19 pandemic. It can provide less biassed and more cost-effective population-level monitoring of the disease burden as compared to clinical testing. An essential component of SARS-CoV-2 wastewater monitoring is next-generation sequencing, providing genomic data to identify and quantify circulating viral strains rapidly. However, the specific choice of sequencing method influences the quality and timeliness of generated data and hence its usefulness for wastewater-based pathogen surveillance. Here, we systematically benchmarked Illumina Novaseq 6000, Element Aviti, ONT R9.4.1 MinION flow cell, and ONT R9.4.1 Flongle flow cell sequencing data to facilitate the selection of sequencing technology. Using a time series of wastewater samples from influent of six wastewater treatment plants throughout Switzerland, along with spike-in experiments, we show that higher sequencing error rates of ONT Nanopore sequencing reduce the accuracy of estimates of the relative abundance of viral variants, but the overall trend is in good concordance among all technologies. We find that the sequencing runtime for ONT Nanopore flow cells can be reduced to as little as five hours without significant impact on the quality of variant estimates. Our findings suggest that SARS-CoV-2 variant tracking is readily achievable with all tested technologies, albeit with different tradeoffs in terms of cost, timeliness and accuracy.

Efficient concentration of viral nucleic acid in wastewater through surfactant releasing and a two-step magnetic bead extraction and purification

Wastewater-based epidemiology (WBE) is a valuable complement to clinical monitoring, allowing for effective surveillance of viral infections in populations, and tracking the presence and the epidemiological dynamics of various infectious pathogens in communities. However, virus loads are usually low-abundant in wastewater, and current virus concentration methods for WBE are laborious and time-consuming with low recovery efficiency. To address these challenges, we have developed a magnetic bead-based semi-automated method involving extraction and purification to directly concentrate viral nucleic acids from sewage within 55 min. Prior to concentration, 0.5 % LDS was introduced to pretreat wastewater to inactivate viruses and release viral nucleic acids from both liquid and solid fractions to improve recovery. Under optimal conditions, the concentration method combined with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA added exogenously in wastewater as low as 4.9 copies/mL within 2.5 h, with an average recovery rate exceeding 80 %. Testing real sewages proved the applicability of the method to detect multiple viruses in different sewages. Additionally, variants of SARS-CoV-2 were successfully identified by multiplex amplicon sequencing in two samples. In conclusion, the new method could provide a much more efficient way for WBE of pathogenic viruses in various sewages.