Wastewater-based Epidemiology Research Articles

Analytical validation of a semi-automated methodology for quantitative measurement of SARS-CoV-2 RNA in wastewater collected in northern New England.

Wastewater-based epidemiology (WBE) can be used to monitor the community presence of infectious disease pathogens of public health concern such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral nucleic acid has been detected in the stool of SARS-CoV-2-infected individuals. Asymptomatic SARS-CoV-2 infections make community monitoring difficult without extensive and continuous population screening. In this study, we validated a procedure that includes manual pre-processing, automated SARS-CoV-2 RNA extraction and detection workflows using both reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR). Genomic RNA and calibration materials were used to create known concentrations of viral material to determine the linearity, accuracy, and precision of the wastewater extraction and SARS-CoV-2 RNA detection. Both RT-qPCR and RT-ddPCR perform similarly in all the validation experiments, with a limit of detection of 50 copies/mL. A wastewater sample from a care facility with a known outbreak was assessed for viral content in replicate, and we showed consistent results across both assays. Finally, in a 2-week survey of two New Hampshire cities, we assessed the suitability of our methods for daily surveillance. This paper describes the technical validation of a molecular assay that can be used for long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.IMPORTANCEThis paper describes the technical validation of a molecular assay that can be used for the long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.

Integrating Wastewater-Based Epidemiology and Mobility Data to Predict SARS-CoV-2 Cases

Wastewater-based epidemiology has garnered considerable research interest, concerning the COVID-19 pandemic. Restrictive public health interventions and mobility limitations are measures to avert a rising case prevalence. The current study integrates WBE monitoring strategies, Google mobility data, and restriction information to assess the epidemiological development of COVID-19. Various SARIMAX models were employed to predict SARS-CoV-2 cases in Liechtenstein and two Austrian regions. This study analyzes four primary strategies for examining the progression of the pandemic waves, described as follows: 1—a univariate model based on active cases; 2—a multivariate model incorporating active cases and WBE data; 3—a multivariate model considering active cases and mobility data; and 4—a sensitivity analysis of WBE and mobility data incorporating restriction policies. Our key discovery reveals that, while WBE for SARS-CoV-2 holds immense potential for monitoring COVID-19 on a societal level, incorporating the analysis of mobility data and restriction policies enhances the precision of the trained models in predicting the state of public health during the pandemic.

Wastewater monitoring of SARS-CoV-2 gene for COVID-19 epidemiological surveillance in Tucumán, Argentina.

Wastewater-based epidemiology provides temporal and spatial information about the health status of a population. The objective of this study was to analyze and report the epidemiological dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the province of Tucumán, Argentina during the second and third waves of coronavirus disease 2019 (COVID-19) between April 2021 and March 2022. The study aimed to quantify SARS-CoV-2 RNA in wastewater, correlating it with clinically reported COVID-19 cases. Wastewater samples (n = 72) were collected from 16 sampling points located in three cities of Tucumán (San Miguel de Tucumán, Yerba Buena y Banda del Río Salí). Detection of viral nucleocapsid markers (N1 gene) was carried out using one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Viral loads were determined for each positive sample using a standard curve. A positive correlation (p < 0.05) was observed between viral load (copies/mL) and the clinically confirmed COVID-19 cases reported at specific sampling points in San Miguel de Tucumán (SP4, SP7, and SP8) in both months, May and June. Indeed, the high viral load concurred with the peaks of COVID-19 cases. This method allowed us to follow the behavior of SARS-CoV-2 infection during epidemic outbreaks. Thus, wastewater monitoring is a valuable epidemiological indicator that enables the anticipation of increases in COVID-19 cases and tracking the progress of the pandemic. SARS-CoV-2 genome-based surveillance should be implemented as a routine practice to prepare for any future surge in infections.

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

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

Optimisation of analytical methods for tuberculosis drug detection in wastewater: A multinational study

Wastewater-based epidemiology (WBE) is a robust tool for disease surveillance and monitoring of pharmaceutical consumption. However, monitoring tuberculosis (TB) drug consumption faces challenges due to limited data availability. This study aimed to optimise methods for detecting TB drugs in treated and untreated wastewater from four African countries: South Africa, Nigeria, Kenya, and Cameroon. The limit of detection (LOD) for these drugs ranged from a minimum of 2.20 (±1.02) for rifampicin to a maximum of 2.95 (±0.79) for pyrazinamide. A parallel trend was observed concerning the limit of quantification (LOQ), with rifampicin reporting the lowest average LOQ of 7.33 (±3.44) and pyrazinamide showing the highest average LOQ of 9.81 (±2.64). The variance in LOD and LOQ values could be attributed to factors such as drug polarity. Erythromycin and rifampicin exhibited moderately polar LogP values (2.6 and 2.95), indicating higher lipid affinity and lower water affinity.Conversely, ethambutol, pyrazinamide, and isoniazid displayed polar LogP values (−0.059, −0.6, and −0.7), suggesting lower lipid affinity and greater water affinity. The study revealed that storing wastewater samples for up to 5 days did not result in significant drug concentration loss, with concentration reduction remaining below 1 log throughout the storage period. Application of the optimised method for drug detection and quantification in both treated and untreated wastewater unveiled varied results. Detection frequencies varied among drugs, with ethambutol consistently most detected, while pyrazinamide and isoniazid were least detected in wastewater from only two countries. Most untreated wastewater samples had undetectable drug concentrations, ranging from 1.21 ng/mL for erythromycin to 54.61 ng/mL for isoniazid. This variability may suggest differences in drug consumption within connected communities. In treated wastewater samples, detectable drug concentrations ranged from 1.27 ng/mL for isoniazid to 10.20 ng/mL for ethambutol. Wastewater treatment plants exhibited variable removal efficiencies for different drugs, emphasising the need for further optimisation. Detecting these drugs in treated wastewater suggests potential surface water contamination and subsequent risks of human exposure, underscoring continued research's importance.

Target and Nontarget Screening to Support Capacity Scaling for Substance Use Assessment through a Statewide Wastewater Surveillance Network in New York.

Wastewater-based epidemiology (WBE) has been widely implemented around the world as a complementary tool to conventional surveillance techniques to inform and improve public health responses. Currently, wastewater surveillance programs in the U.S. are evaluating integrated approaches to address public health challenges across multiple domains, including substance abuse. In this work, we demonstrated the potential of online solid-phase extraction coupled with liquid chromatography-high-resolution mass spectrometry to support targeted quantification and nontargeted analysis of psychoactive and lifestyle substances as a step toward understanding the operational feasibility of a statewide wastewater surveillance program for substance use assessment in New York. Target screening confirmed 39 substances in influent samples collected from 10 wastewater treatment plants with varying sewershed characteristics and is anticipated to meet the throughput demands as the statewide program scales up to full capacity. Nontarget screening prioritized additional compounds for identification at three confidence levels, including psychoactive substances, such as opioid analgesics, phenethylamines, and cathinone derivatives. Consumption rates of 12 target substances detected in over 80% of wastewater samples were similar to those reported by previous U.S.-based WBE studies despite the uncertainty associated with back-calculations. For selected substances, the relative bias in consumption estimates was sensitive to variations in monitoring frequency, and factors beyond human excretion (e.g., as indicated by the parent-to-metabolite ratios) might also contribute to their prevalence at the sewershed scale. Overall, our study marks the initial phase of refining analytical workflows and data interpretation in preparation for the incorporation of substance use assessment into the statewide wastewater surveillance program in New York.

Bacterial community profiles within the water samples of leptospirosis outbreak areas.

Leptospirosis is a water-related zoonotic disease. The disease is primarily transmitted from animals to humans through pathogenic Leptospira bacteria in contaminated water and soil. Rivers have a critical role in Leptospira transmissions, while co-infection potentials with other waterborne bacteria might increase the severity and death risk of the disease. The water samples evaluated in this study were collected from four recreational forest rivers, Sungai Congkak, Sungai Lopo, Hulu Perdik, and Gunung Nuang. The samples were subjected to next-generation sequencing (NGS) for the 16S rRNA and in-depth metagenomic analysis of the bacterial communities. The water samples recorded various bacterial diversity. The samples from the Hulu Perdik and Sungai Lopo downstream sampling sites had a more significant diversity, followed by Sungai Congkak. Conversely, the upstream samples from Gunung Nuang exhibited the lowest bacterial diversity. Proteobacteria, Firmicutes, and Acidobacteria were the dominant phyla detected in downstream areas. Potential pathogenic bacteria belonging to the genera Burkholderiales and Serratia were also identified, raising concerns about co-infection possibilities. Nevertheless, Leptospira pathogenic bacteria were absent from all sites, which is attributable to its limited persistence. The bacteria might also be washed to other locations, contributing to the reduced environmental bacterial load. The present study established the presence of pathogenic bacteria in the river ecosystems assessed. The findings offer valuable insights for designing strategies for preventing pathogenic bacteria environmental contamination and managing leptospirosis co-infections with other human diseases. Furthermore, closely monitoring water sample compositions with diverse approaches, including sentinel programs, wastewater-based epidemiology, and clinical surveillance, enables disease transmission and outbreak early detections. The data also provides valuable information for suitable treatments and long-term strategies for combating infectious diseases.

Leveraging wastewater surveillance for managing the spread of SARS-CoV-2 and concerned pathogens during FIFA World Cup Qatar 2022

Wastewater-based epidemiology (WBE) has been proven effective for the monitoring of infectious disease outbreaks during mass gathering events and for timely public health interventions. As part of Qatar's efforts to monitor and combat the spread of infectious diseases during the FIFA World Cup Qatar 2022™ (FWC’22), wastewater surveillance was used to monitor the spread of SARS-CoV-2, human enterovirus, and poliovirus. The screening covered five major wastewater treatment plants servicing the event locations between October 2022 and January 2023. Viruses were concentrated from the wastewater samples by PEG precipitation, followed by qRT-PCR to measure the viral load in the wastewater. As expected, SARS-CoV-2 and enterovirus RNA were detected in all samples, while poliovirus was not detected. The concentration of SARS-CoV-2 was correlated with population density, such as areas surrounding the World Cup venues, and with the number of reported clinical cases. Additionally, we observed temporal fluctuations in viral RNA concentrations, with peak levels coinciding with the group stage matches of the FWC’22. This study has been useful in providing public health authorities with an efficient and cost-effective surveillance system for potential infectious disease outbreaks during mega-events.

Precision and Accuracy Limits of Wastewater-Based Epidemiology—Lessons Learned from SARS-CoV-2: A Scoping Review

Background: Wastewater-based epidemiology (WBE) has become crucial for early microbial outbreak detection and public health surveillance globally, underscored by the COVID-19 pandemic. However, despite advancements in sampling and analyses, interpreting results and estimating infection rates pose challenges. Enhancements in sewer system engineering, understanding the wastewater environment, and addressing the impact of the environment on the accuracy of results are needed. Objective: This scoping review aims to identify engineering knowledge gaps in WBE to guide future study designs. Design: Research on “wastewater-based epidemiology” involving “engineering”, published between 2015 and 2023, was extracted from the Scopus database. Results: This scoping review examines elements influencing WBE’s precision and reliability, especially in identifying and measuring SARS-CoV-2 RNA. It identifies significant effects of engineering, analytical practices, and the wastewater’s composition on WBE performance. Conclusions: This review calls for further investigation into economical evaluation methods of these factors to enhance WBE data normalization and interpretation, utilizing existing wastewater treatment plant data used for treatment control, which could be a cost-effective approach over more expensive population biomarkers. This approach, aside from SARS-CoV-2, holds potential for application to a broader number and types of diseases, as well as population consumption habits.

Wastewater-based epidemiology as a public health resource in low- and middle-income settings

In the face of emerging and re-emerging diseases, novel and innovative approaches to population scale surveillance are necessary for the early detection and quantification of pathogens. The last decade has seen the rapid development of wastewater and environmental surveillance (WES) to address public health challenges, which has led to establishment of wastewater-based epidemiology (WBE) approaches being deployed to monitor a range of health hazards. WBE exploits the fact that excretions and secretions from urine, and from the gut are discharged in wastewater, particularly sewage, such that sampling sewage systems provides an early warning system for disease outbreaks by providing an early indication of pathogen circulation. While WBE has been mainly used in locations with networked wastewater systems, here we consider its value for less connected populations typical of lower-income settings, and in assess the opportunity afforded by pit latrines to sample communities and localities. We propose that where populations struggle to access health and diagnostic facilities, and despite several additional challenges, sampling unconnected wastewater systems remains an important means to monitor the health of large populations in a relatively cost-effective manner.

A systematic review on the incidence of influenza viruses in wastewater matrices: Implications for public health.

Influenza viruses pose a significant public health threat, necessitating comprehensive surveillance strategies to enhance early detection and preventive measures. This systematic review investigates the incidence of influenza viruses in wastewater matrices, aiming to elucidate the potential implications for public health. The study synthesizes existing literature, employing rigorous inclusion criteria to identify relevant studies conducted globally. The essence of the problem lies in the gaps of traditional surveillance methods, which often rely on clinical data and may underestimate the true prevalence of influenza within communities. Wastewater-based epidemiology offers a novel approach to supplementing these conventional methods, providing a broader and more representative assessment of viral circulation. This review systematically examines the methodologies employed in the selected studies, including virus concentration techniques and molecular detection methods, to establish a standardized framework for future research. Our findings reveal a consistent presence of influenza viruses in diverse wastewater matrices across different geographic locations and seasons. Recommendations for future research include the standardization of sampling protocols, improvement of virus concentration methods, and the integration of wastewater surveillance into existing public health frameworks. In conclusion, this systematic review contributes to the understanding of influenza dynamics in wastewater matrices, offering valuable insights for public health practitioners and policymakers. Implementation of wastewater surveillance alongside traditional methods can enhance the resilience of public health systems and better prepare communities for the challenges posed by influenza outbreaks.

Wastewater tiling amplicon sequencing in sentinel sites reveals longitudinal dynamics of SARS-CoV-2 variants prevalence

The ongoing evolution of SARS-CoV-2 is a significant concern, especially with the decrease in clinical sequencing efforts, which impedes the ability of public health sectors to prepare for the emergence of new variants and potential COVID-19 outbreaks. Wastewater-based epidemiology (WBE) has been proposed as a surveillance program to detect and monitor the SARS-CoV-2 variants being transmitted in communities. However, research is limited in evaluating the effectiveness of wastewater collection at sentinel sites for monitoring disease prevalence and variant dynamics, especially in terms of inferring the epidemic patterns on a broader scale, such as at the state/province level. This study utilized a multiplexed tiling amplicon-based sequencing (ATOPlex) to track the longitudinal dynamics of variant of concern (VOC) in wastewater collected from municipalities in Queensland, Australia, spanning from 2020 to 2022. We demonstrated that wastewater epidemiology measured by ATOPlex exhibited a strong and consistent correlation with the number of daily confirmed cases. The VOC dynamics observed in wastewater closely aligned with the dynamic profile reported by clinical sequencing. Wastewater sequencing has the potential to provide early warning information for emerging variants. These findings suggest that WBE at sentinel sites, coupled with sensitive sequencing methods, provides a reliable and long-term disease surveillance strategy.

Occurrence of micropollutants in rural domestic wastewater in Zhejiang Province, China and corresponding wastewater-based epidemiology analysis

By 2021, rural regions in China were occupied by over 500 million residents, generating an annual volume of 19.5 billion m3 of rural domestic wastewater (RDW). This study aimed to investigate the occurrence and removal of micropollutants (MPs) in RDW treatment facilities and to perform a corresponding wastewater-based epidemiology analysis (WBE). Our findings indicated the significantly high levels of influent MPs, particularly pharmaceuticals, such as ofloxacin and diclofenac being most prevalent (ranging from several to tens of μg/L) across different facilities. After various treatments, regular water indexes in the effluent, like NH3 -N and COD, have basically satisfied the local discharge standard. However, the concentration of certain dominant MPs in effluent remained notably high, ranging from hundreds of ng/L to several μg/L. The risk quotients of MPs like diclofenac, ciprofloxacin, ofloxacin, sulfamethoxazole, diuron, and isoproturon were all above 1 in the effluent, signifying significant hazards to aquatic organisms. The quantitative meta-analysis revealed higher average standardized removal efficiency for membrane bioreactor (MBR) treatment (-11 %) compared to anaerobic/anoxic/aerobic (A2O) treatment (11 %), indicating the higher efficiency of MBR treatment in outperforming the A2O as a secondary treatment. Additionally, employing biofilter as a tertiary treatment proved to be more effective as compared to flocculation-air flotation and artificial wetlands. Moreover, the results of WBE analysis showed that diclofenac and ofloxacin emerged as the most commonly used pharmaceuticals (of seven), with consumption levels recorded at 1222 and 517 mg/(d·103 capita), with daily defined doses per day per 103 capita of 12.2/1000 and 1.29/1000, respectively. This study addresses the existing knowledge gaps regarding the occurrence and removal of MPs in RDW and offers valuable insights into pharmaceutical consumption patterns in rural regions, thereby improving our understanding of public health.

Antimicrobials and antimicrobial resistance genes in the shadow of COVID-19 pandemic: A wastewater-based epidemiology perspective

Higher usage of antimicrobial agents in both healthcare facilities and the communities has resulted in an increased spread of resistant bacteria. However, the improved infection prevention and control practices may also contribute to decreasing antimicrobial resistance (AMR). In the present study, wastewater-based epidemiology (WBE) approach was applied to explore the link between COVID-19 and the community usage of antimicrobials, as well as the prevalence of resistance genes. Longitudinal study has been conducted to monitor the levels of 50 antimicrobial agents (AAs), 24 metabolites, 5 antibiotic resistance genes (ARGs) and class 1 integrons (intI 1) in wastewater influents in 4 towns/cities over two years (April 2020 - March 2022) in the South-West of England (a total of 1,180 samples collected with 87,320 individual AA measurements and 8,148 ARG measurements). Results suggested higher loads of AAs and ARGs in 2021–22 than 2020–21, with beta-lactams, quinolones, macrolides and most ARGs showing statistical differences. In particular, the intI 1 gene (a proxy of environmental ARG pollution) showed a significant increase after the ease of the third national lockdown in England. Positive correlations for all quantifiable parent AAs and metabolites were observed, and consumption vs direct disposal of unused AAs has been identified via WBE. This work can help establish baselines for AMR status in communities, providing community-wide surveillance and evidence for informing public health interventions. Overall, studies focused on AMR from the start of the pandemic to the present, especially in the context of environmental settings, are of great importance to further understand the long-term impact of the pandemic on AMR.

Comparative Inactivation of the RNA of the Delta and Omicron Variants of SARS-CoV-2 in Wastewater of Five Municipalities in Southeast Germany

During the COVID-19 pandemic, wastewater-based epidemiology has been extensively used as a helpful tool for evaluation of the epidemic situation in catchments of wastewater treatment plants. In this context, knowledge about the rate of virus inactivation in wastewater is important for characterization of the influence of retention times in sewers on virus concentrations, of the storage periods before analysis in the laboratory, on virus elimination during treatment and for modeling approaches. In the present study, we genotyped SARS-CoV-2 in the respiratory tract specimens of infected patients and spiked the raw wastewater of five treatment plants with aliquots of these samples. The test preparations were incubated under practically relevant temperature conditions (14 °C) over 31 days. The linear decay rates resulted in comparable T90 values (30.5 d) for the Delta and the Omicron BA1 variants of SARS-CoV-2 without differences in using the E or S gene of the virus as the target for amplification. In contrast, the origin of wastewater influenced the inactivation rate of both variants significantly, with the mean T90 values varying between 24.3 and 53.1 days in the wastewater from the five plants. The data suggest that the inactivation rate of SARS-CoV-2 in wastewater is more strongly determined by the composition of the water than by the virus variant.

Genomic Analysis and Surveillance of Respiratory Syncytial Virus (RSV) Using Wastewater-Based Epidemiology (WBE).

Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limits the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of SARS-CoV-2 in the community but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying RT-qPCR and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and non-pharmaceutical interventions.

Efficient magnetic solid-phase extraction, UPLC-MS/MS detection, and consumption assessment of five trace psychoactive substances.

Wastewater-based epidemiology (WBE) has become an objective and updated surveillance strategy for monitoring and estimating consumption trends of psychoactive substances (PSs) in the population. Firstly, magnetic shrimp shell biochar-based adsorbent (DZMBC) was synthesized and employed for extraction trace PSs from municipal wastewater. Proper pyrolysis temperature and increased KOH activator content favored the pore structure and surface area, thus facilitating extraction. DZMBC delivered exceptional extraction performance such as pH stability, anti-interference property, fast magnetic separation ability, reusability, and reproducibility. Then, a method based on magnetic solid-phase extraction (MSPE) followed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed, validated, and utilized for the quantitative determination of five PSs in real wastewater samples. Methodological validation results indicated a favorable linearity, low method limits of detection (1.00-4.75ng/L), and good precisions (intra-day and inter-day relative standard deviations < 4.8%). Finally, an objective snapshot of Chongqing drug use and consumption pattern was obtained. Methamphetamine (MAMP) and 3,4-methylenedioxymethamphetamine (MDMA) were the prevalent illegal drugs in local. Both concentrations and per capita consumption of MDMA displayed a change (P < 0.05) between July and September, while no statistical differences were observed for each week.

Determination of 9 fentanyl drugs in environmental water by UHPLC-MS/MS combined with solid phase extraction

In this study, sensitive and selective SPE-UHPLC-MS/MS methods were established and applied to the determination of 9 fentanyl drugs in environmental water, in order to address the lack of research on wastewater-based epidemiology for fentanyl drugs. The methods involved purifying and enriching water samples using homemade PS/DVB-WCX SPE cartridges, followed by UHPLC-MS/MS analysis. In subsequent research, the performance of four SPE cartridges were compared, and the results revealed the superior performance of PS/DVB-WCX in terms of extraction efficiency and matrix removal. Optimized instrument parameters enabled qualitative and quantitative analysis of the 9 fentanyl drugs. For different matrices, two quantitative methods were developed and their correlation coefficient (r2) were all greater than 0.9945. The LODs and LOQs of all analytes were 0.17 ng/L and 0.50 ng/L respectively. Accuracy assessment via spiking standards into environmental water samples showed recoveries ranging from 81.4 % to 109.4 % and 81.6 % to 116.7 % respectively for two methods. The results suggested that the proposed methods were able to meet the needs of 9 fentanyl drugs monitoring in environmental water. Additionally, the greenness assessment of the proposed analytical method was evaluated by three greenness assessment metrics: National environmental methods Index (NEMI), Green analytical procedure Index (GAPI), and Analytical GREEnness metric (AGREE). The results indicate that the established method is environmentally friendly.

High-resolution and real-time wastewater viral surveillance by Nanopore sequencing

Wastewater genomic sequencing stands as a pivotal complementary tool for viral surveillance in populations. While long-read Nanopore sequencing is a promising platform to provide real-time genomic data, concerns over the sequencing accuracy of the earlier Nanopore versions have somewhat restrained its widespread application in wastewater analysis. Here, we evaluate the latest improved version of Nanopore sequencing (R10.4.1), using SARS-CoV-2 as the model infectious virus, to demonstrate its effectiveness in wastewater viral monitoring. By comparing amplicon lengths of 400 bp and 1200 bp, we revealed that shorter PCR amplification is more suitable for wastewater samples due to viral genome fragmentation. Utilizing mock wastewater samples, we validated the reliability of Nanopore sequencing for variant identification by comparing it with Illumina sequencing results. The strength of Nanopore sequencing in generating real-time genomic data for providing early warning signals was also showcased, indicating that as little as 0.001 Gb of data can provide accurate results for variant prevalence. Our evaluation also identified optimal alteration frequency cutoffs (>50 %) for precise mutation profiling, achieving >99 % precision in detecting single nucleotide variants (SNVs) and insertions/deletions (indels). Monitoring two major wastewater treatment plants in Hong Kong from September 2022 to April 2023, covering over 4.5 million population, we observed a transition in dominant variants from BA.5 to XBB lineages, with XBB.1.5 being the most prevalent variants. Mutation detection also highlighted the potential of wastewater Nanopore sequencing in uncovering novel mutations and revealed links between signature mutations and specific variants. This study not only reveals the environmental implications of Nanopore sequencing in SARS-CoV-2 surveillance but also underscores its potential in broader applications including environmental health monitoring of other epidemic viruses, which could significantly enhance the field of wastewater-based epidemiology.

Group A Streptococcus pyogenes in wastewater: Applicability of wastewater-based epidemiology for monitoring the prevalence of GAS pharyngitis during the late COVID-19 pandemic phase

Streptococcus pyogenes, Group A Streptococcus (GAS), is a human pathogen that causes a spectrum of diseases from mild to severe, including GAS pharyngitis, a common acute respiratory disease in developed countries. Although wastewater-based epidemiology (WBE) has been extensively used to monitor viral pathogens such as severe acute respiratory syndrome coronavirus 2, its applicability to S. pyogenes remains unexplored. This study was conducted to investigate the feasibility of detecting and quantifying S. pyogenes in wastewater by quantitative polymerase chain reaction (qPCR) and evaluate the applicability of WBE for monitoring the prevalence of GAS pharyngitis. A total of 52 grab influent samples were collected from a wastewater treatment plant in Japan once a week between March 2023 and February 2024. The samples were centrifuged, followed by nucleic acid extraction and qPCR for the S. pyogenes-specific genes speB and spy1258. Of the 52 samples, 90 % and 81 % were positive for speB and spy1258 genes, respectively, indicating the feasibility of S. pyogenes for wastewater surveillance. However, the percentage of quantifiable samples for speB gene was significantly higher in winter than in spring and summer. Similarly, the concentrations of both genes in wastewater samples were significantly higher in winter (speB, 4.1 ± 0.27 log10 copies/L; spy1258, 4.1 ± 0.28 log10 copies/L; One-way ANOVA, p < 0.01) than in spring and summer. Higher concentrations and detection ratios of S. pyogenes genes were observed during increased GAS pharyngitis cases in the catchment. Significant moderate correlations were observed between target gene concentrations and reported GAS pharyngitis cases. This study enhances the understanding role of WBE in monitoring and managing infectious diseases within communities.