Surveillance of SARS-CoV-2 Employing Wastewater

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Public Health Microbiology".

Deadline for manuscript submissions: 30 June 2025 | Viewed by 13500

Special Issue Editor


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Guest Editor
European Commission, Joint Research Centre (JRC), 21027 Ispra, Italy
Interests: wastewater and environmental surveillance (WES); SARS-CoV-2; public health; COVID-19 pandemic; pathogen monitoring

Special Issue Information

Dear Colleagues,

This Special Issue, entitled "Surveillance of SARS-CoV-2 Employing Wastewater", chronicles the evolution of Wastewater and Environmental Surveillance (WES) from the COVID-19 pandemic into 2024. As SARS-CoV-2 has become endemic, WES has risen as the primary form of viral tracking in many countries, underscoring its importance in public health. As of 2024, WES advancements have enhanced the detection of SARS-CoV-2 and contributed to the monitoring of other pathogens, illustrating its potential for widespread use in public health.

Developments in WES have enabled public health officials in many countries to proactively manage community infection levels with increased sensitivity and specificity. This Special Issue emphasizes the advancement of health sector leadership, ethical practices, and integration with public health surveillance. It delves into the tactical aspects of wastewater surveillance, including sampling, storage, laboratory analysis, data normalization, and communication.

This Special Issue serves to synethize the knowledge gained from the pandemic, demonstrating the application of WES in a One-Health context and its expanding role in the detection and management of infectious diseases. WES is becoming a key element in global health security, playing an active  role in the early detection of health threats.

Dr. Bernd Manfred Gawlik
Guest Editor

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Keywords

  • wastewater and environmental surveillance (WES)
  • SARS-CoV-2
  • public health
  • COVID-19 pandemic
  • pathogen monitoring
  • global health security

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Published Papers (9 papers)

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22 pages, 4509 KiB  
Article
Wastewater Speaks: Evaluating SARS-CoV-2 Surveillance, Sampling Methods, and Seasonal Infection Trends on a University Campus
by Shilpi Bhatia, Tinyiko Nicole Maswanganye, Olusola Jeje, Danielle Winston, Mehdi Lamssali, Dongyang Deng, Ivory Blakley, Anthony A. Fodor and Liesl Jeffers-Francis
Microorganisms 2025, 13(4), 924; https://doi.org/10.3390/microorganisms13040924 - 17 Apr 2025
Viewed by 235
Abstract
Wastewater surveillance has emerged as a cost-effective and equitable approach for tracking the spread of SARS-CoV-2. In this study, we monitored the prevalence of SARS-CoV-2 on a university campus over three years (2021–2023) using wastewater-based epidemiology (WBE). Wastewater samples were collected from 11 [...] Read more.
Wastewater surveillance has emerged as a cost-effective and equitable approach for tracking the spread of SARS-CoV-2. In this study, we monitored the prevalence of SARS-CoV-2 on a university campus over three years (2021–2023) using wastewater-based epidemiology (WBE). Wastewater samples were collected from 11 manholes on campus, each draining wastewater from a corresponding dormitory building, and viral RNA concentrations were measured using reverse transcription-quantitative PCR (RT-qPCR). Weekly clinical case data were also obtained from the university health center. A strong positive and significant correlation was observed between Grab and Composite sampling methods, supporting their robustness as equally effective approaches for sample collection. Specifically, a strong correlation was observed between Aggie Village 4 Grab and Aggie Village 4 Composite samples (R2 = 0.84, p = 0.00) and between Barbee Grab and Barbee Composite samples (R2 = 0.80, p = 0.00). Additionally, higher viral RNA copies of SARS-CoV-2 (N1 gene) were detected during the Spring semester compared to the Fall and Summer semesters. Notably, elevations in raw N1 concentrations were observed shortly after the return of college students to campus, suggesting that these increases were predominantly associated with students returning at the beginning of the Fall and Spring semesters (January and August). To account for variations in fecal loading, SARS-CoV-2 RNA concentrations were normalized using Pepper Mild Mottle Virus (PMMoV), a widely used viral fecal biomarker. However, normalization using PMMoV did not improve correlations between SARS-CoV-2 RNA levels and clinical case data. Despite these findings, our study did not establish WBE as a consistently reliable complement to clinical testing in a university campus setting, contrary to many retrospective studies. One key limitation was that numerous off-campus students did not contribute to the campus wastewater system corresponding to the monitored dormitories. However, some off-campus students were still subjected to clinical testing at the university health center under mandated protocols. Moreover, the university health center discontinued reporting cases per dormitory after 2021, making direct comparisons more challenging. Nevertheless, this study highlights the continued value of WBE as a surveillance tool for monitoring infectious diseases and provides critical insights into its application in campus environments. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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20 pages, 2789 KiB  
Article
Evaluating Interlaboratory Variability in Wastewater-Based COVID-19 Surveillance
by Arianna Azzellino, Laura Pellegrinelli, Ramon Pedrini, Andrea Turolla, Barbara Bertasi, Sandro Binda, Sara Castiglioni, Clementina E. Cocuzza, Fabio Ferrari, Andrea Franzetti, Maria Giovanna Guiso, Marina Nadia Losio, Marianna Martinelli, Antonino Martines, Rosario Musumeci, Desdemona Oliva, Laura Sandri, Valeria Primache, Francesco Righi, Annalisa Scarazzato, Silvia Schiarea, Elena Pariani, Emanuela Ammoni, Danilo Cereda and Francesca Malpeiadd Show full author list remove Hide full author list
Microorganisms 2025, 13(3), 526; https://doi.org/10.3390/microorganisms13030526 - 27 Feb 2025
Viewed by 708
Abstract
Wastewater-based environmental surveillance enables the monitoring of SARS-CoV-2 dynamics within populations, offering critical epidemiological insights. Numerous workflows for tracking SARS-CoV-2 have been developed globally, underscoring the need for interlaboratory comparisons to ensure data consistency and comparability. An inter-calibration test was conducted among laboratories [...] Read more.
Wastewater-based environmental surveillance enables the monitoring of SARS-CoV-2 dynamics within populations, offering critical epidemiological insights. Numerous workflows for tracking SARS-CoV-2 have been developed globally, underscoring the need for interlaboratory comparisons to ensure data consistency and comparability. An inter-calibration test was conducted among laboratories within the network monitoring SARS-CoV-2 in wastewater samples across the Lombardy region (Italy). The test aimed to evaluate data reliability and identify potential sources of variability using robust statistical approaches. Three wastewater samples were analyzed in parallel by four laboratories using identical pre-analytical (PEG-8000-based centrifugation) and analytical processes (qPCR targeting N1/N3 and Orf-1ab). A two-way ANOVA framework within Generalized Linear Models was applied, and multiple pairwise comparisons among laboratories were performed using the Bonferroni post hoc test. The statistical analysis revealed that the primary source of variability in the results was associated with the analytical phase. This variability was likely influenced by differences in the standard curves used by the laboratories to quantify SARS-CoV-2 concentrations, as well as the size of the wastewater treatment plants. The findings of this study highlight the importance of interlaboratory testing in verifying the consistency of analytical determinations and in identifying the key sources of variation. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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17 pages, 3549 KiB  
Article
Wastewater-Based Epidemiological Surveillance in France: The SUM’EAU Network
by Frédéric Jourdain, Laila Toro, Zoé Senta-Loÿs, Marilyne Deryene, Walid Mokni, Tess Azevedo Da Graça, Yann Le Strat, Sofiane Rahali, Ami Yamada, Anna Maisa, Maël Pretet, Jeanne Sudour, Christophe Cordevant, Thierry Chesnot, Veronica Roman, Amandine Wilhelm, Benoît Gassilloud and Damien Mouly
Microorganisms 2025, 13(2), 281; https://doi.org/10.3390/microorganisms13020281 - 26 Jan 2025
Cited by 1 | Viewed by 1566
Abstract
Wastewater surveillance is a powerful public health tool which gained global prominence during the COVID-19 pandemic. This article describes the development and implementation of the national wastewater surveillance network in France: SUM’EAU. Preliminary work included defining a sampling strategy, evaluating/optimising analytical methods, launching [...] Read more.
Wastewater surveillance is a powerful public health tool which gained global prominence during the COVID-19 pandemic. This article describes the development and implementation of the national wastewater surveillance network in France: SUM’EAU. Preliminary work included defining a sampling strategy, evaluating/optimising analytical methods, launching a call for tenders to select network laboratories and producing wastewater monitoring indicators. SUM’EAU was then deployed in three stages: (i) a pilot study, (ii) the transfer of analytical activities from the National Reference Laboratory to four selected network laboratories, and (iii) the extension of the system to additional sampling sites. Currently, SUM’EAU monitors SARS-CoV-2 across 54 wastewater treatment plants in mainland France. Once a week on business days, 24 h flow-proportional composite samples are collected at plant inlets and transported at 5 °C (±3 °C) to partner laboratories for analysis. The analytical process involves sample concentration, RNA extraction, and digital RT-PCR/q-RT-PCR to detect and quantify the presence of the SARS-CoV-2 genome in wastewater. Subsequently, data are transferred to Santé publique France, the French National Public Health Agency, for analysis and interpretation. While SUM’EAU has been instrumental in monitoring the COVID-19 pandemic and holds significant potential for broader application, securing sustainable funding for its operation remains a major challenge. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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19 pages, 3642 KiB  
Article
Wastewater Surveillance for SARS-CoV-2 in Northern Italy: An Evaluation of Three Different Gene Targets
by Giulio Mannarà, Marianna Martinelli, Chiara Giubbi, Michelle Rizza, Eleonora Giordano, Federica Perdoni, Erika Bruno, Annalisa Morella, Arianna Azzellino, Andrea Turolla, Ramon Pedrini, Francesca Malpei, Giuseppina La Rosa, Elisabetta Suffredini, Danilo Cereda, Emanuela Ammoni, Simone Villa, Francesca Pregnolato, Marialuisa Lavitrano, Andrea Franzetti, Rosario Musumeci and Clementina E. Cocuzzaadd Show full author list remove Hide full author list
Microorganisms 2025, 13(2), 236; https://doi.org/10.3390/microorganisms13020236 - 22 Jan 2025
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Abstract
Wastewater-based epidemiology has emerged as a complementary tool for the monitoring of COVID-19 pandemic waves and for the circulation of viral variants. The selection, standardization, and dynamics of different SARS-CoV-2 RNA targets in wastewater requires further investigation. In the present study, 106 wastewater [...] Read more.
Wastewater-based epidemiology has emerged as a complementary tool for the monitoring of COVID-19 pandemic waves and for the circulation of viral variants. The selection, standardization, and dynamics of different SARS-CoV-2 RNA targets in wastewater requires further investigation. In the present study, 106 wastewater samples were collected over a 24-month period from the wastewater treatment plant of Sondrio, north Italy, and were analyzed for the presence of SARS-CoV-2 RNA through the quantification of ORF1b, N1, and N3 gene targets via one-step real-time qPCR. In general, the three RNA targets demonstrated different performances and dynamics over the studied time period, underlying the usefulness of multiple viral targets in the surveillance of SARS-CoV-2 in wastewater. During the first 12 months, the quantification of the selected SARS-CoV-2 viral targets also correlated with the reported clinical cases in the same geographical area; however, from the overall data analysis this did not appear to significantly anticipate the epidemic waves. In conclusion, this study further supports the use of wastewater surveillance as a real time indicator of the human circulation of SARS-CoV-2. Moreover, the use of multiple viral gene targets has been shown to improve the reliability of SARS-CoV-2 surveillance in wastewater over time. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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17 pages, 2806 KiB  
Article
The Impact of Viral Concentration Method on Quantification and Long Amplicon Nanopore Sequencing of SARS-CoV-2 and Noroviruses in Wastewater
by George Scott, Nicholas P. Evens, Jonathan Porter and David I. Walker
Microorganisms 2025, 13(2), 229; https://doi.org/10.3390/microorganisms13020229 - 22 Jan 2025
Viewed by 952
Abstract
Wastewater-based surveillance has gained attention in the four years following the start of the COVID-19 pandemic. Accurate pathogen detection, quantification and characterisation rely on the selection of appropriate methodologies. Here, we explore the impact of viral concentration method on RT-qPCR inhibition and quantification [...] Read more.
Wastewater-based surveillance has gained attention in the four years following the start of the COVID-19 pandemic. Accurate pathogen detection, quantification and characterisation rely on the selection of appropriate methodologies. Here, we explore the impact of viral concentration method on RT-qPCR inhibition and quantification of norovirus genogroups I and II (GI and GII), crAssphage, phi6 and SARS-CoV-2. Additionally, their impact on long amplicon sequencing for typing noroviruses and whole-genome sequencing (WGS) SARS-CoV-2 was explored. RT-qPCR inhibition for each viral concentration method was significantly different apart from the two ultrafiltration methods, InnovaPrep® concentrating pipette (IP) and Vivaspin® (VS) centrifugal concentrators. Using an ultrafiltration method reduced inhibition by 62.0% to 96.0% compared to the ammonium sulphate (AS) and polyethylene glycol (PEG) precipitation-based methods. Viral quantification was significantly impacted by concentration method with the highest concentrations (copies/L) observed for VS with 7.2- to 83.2-fold differences from AS depending on the target. Norovirus long amplicon sequencing showed genotype-dependent differences with IP performing best for GI and VS for GII although IP performance gains for GI were relatively small. VS outperformed AS and IP across all metrics during SARS-CoV-2 WGS. Overall, VS performed the best when considering all the areas of investigation. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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17 pages, 4373 KiB  
Article
Effective Inhibitor Removal from Wastewater Samples Increases Sensitivity of RT-dPCR and Sequencing Analyses and Enhances the Stability of Wastewater-Based Surveillance
by Nico Linzner, Alexander Bartel, Vera Schumacher, José Horacio Grau, Emanuel Wyler, Henrike Preuß, Sonja Garske, Julia Bitzegeio, Elisabeth Barbara Kirst, Karsten Liere, Sebastian Hoppe, Tatiana A. Borodina, Janine Altmüller, Markus Landthaler, Martin Meixner, Daniel Sagebiel and Uta Böckelmann
Microorganisms 2024, 12(12), 2475; https://doi.org/10.3390/microorganisms12122475 - 2 Dec 2024
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Abstract
Wastewater-based surveillance (WBS) is a proven tool for monitoring population-level infection events. Wastewater contains high concentrations of inhibitors, which contaminate the total nucleic acids (TNA) extracted from these samples. We found that TNA extracts from raw influent of Berlin wastewater treatment plants contained [...] Read more.
Wastewater-based surveillance (WBS) is a proven tool for monitoring population-level infection events. Wastewater contains high concentrations of inhibitors, which contaminate the total nucleic acids (TNA) extracted from these samples. We found that TNA extracts from raw influent of Berlin wastewater treatment plants contained highly variable amounts of inhibitors that impaired molecular analyses like dPCR and next-generation sequencing (NGS). By using dilutions, we were able to detect inhibitory effects. To enhance WBS sensitivity and stability, we applied a combination of PCR inhibitor removal and TNA dilution (PIR+D). This approach led to a 26-fold increase in measured SARS-CoV-2 concentrations, practically reducing the detection limit. Additionally, we observed a substantial increase in the stability of the time series. We define suitable stability as a mean absolute error (MAE) below 0.1 log10 copies/L and a geometric mean relative absolute error (GMRAE) below 26%. Using PIR+D, the MAE could be reduced from 0.219 to 0.097 and the GMRAE from 65.5% to 26.0%, and even further in real-world WBS. Furthermore, PIR+D improved SARS-CoV-2 genome alignment and coverage in amplicon-based NGS for low to medium concentrations. In conclusion, we strongly recommend both the monitoring and removal of inhibitors from samples for WBS. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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10 pages, 3594 KiB  
Communication
One Year of Wastewater Surveillance in South Africa Supporting COVID-19 Clinical Findings Across Two Waves of Infection
by Renée Street, Angela Mathee, Tarylee Reddy, Nomfundo T. Mahlangeni, Noluxabiso Mangwana, Sizwe Nkambule, Candice Webster, Stephanie Dias, Jyoti Rajan Sharma, Pritika Ramharack, Johan Louw, Swastika Surujlal-Naicker, Natacha Berkowitz, Mongezi Mdhluli, Glenda Gray, Christo Muller and Rabia Johnson
Microorganisms 2024, 12(11), 2230; https://doi.org/10.3390/microorganisms12112230 - 4 Nov 2024
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Abstract
Wastewater-based epidemiology (WBE) has been an important tool for the detection of COVID-19 outbreaks. The retrospective analysis of COVID-19 data is vital to understand the spread and impact of the virus as well as to inform future planning and response efforts. In this [...] Read more.
Wastewater-based epidemiology (WBE) has been an important tool for the detection of COVID-19 outbreaks. The retrospective analysis of COVID-19 data is vital to understand the spread and impact of the virus as well as to inform future planning and response efforts. In this study, we evaluated the SARS-CoV-2 RNA levels in wastewater from 21 wastewater treatment plants (WWTPs) in the City of Cape Town (South Africa) over a period of 12 months and compared the (inactive) SARS-CoV-2 viral RNA in wastewater between wave 2 (November 2020 to January 2021) and wave 3 (June 2021 to September 2021). The SARS-CoV-2 RNA expression was quantified in wastewater using quantitative real-time PCR (qRT-PCR) by targeting the nucleocapsid (N) gene, and the resultant signal was normalized to the WWTP design capacity and catchment size. Our findings show that the maximum SARS-CoV-2 RNA signal was significantly higher in wave 3 than in wave 2 (p < 0.01). The duration of wave 3 (15 weeks) was longer than that of wave 2 (10 weeks), and the wastewater surveillance data supported the clinical findings, as evidenced by the two distinct waves. Furthermore, the data demonstrated the importance of long-term wastewater surveillance as a key indicator of changing trends. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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16 pages, 3161 KiB  
Article
Wastewater Surveillance of SARS-CoV-2 in Slovenia: Key Public Health Tool in Endemic Time of COVID-19
by Natalija Kranjec, Andrej Steyer, Tjaša Cerar Kišek, Tom Koritnik, Tea Janko, Maja Bolješić, Vid Vedlin, Verica Mioč, Barbara Lasecky, Tatjana Jurša, José Gonçalves, Herbert Oberacher, Alenka Trop Skaza, Mario Fafangel and An Galičič
Microorganisms 2024, 12(11), 2174; https://doi.org/10.3390/microorganisms12112174 - 29 Oct 2024
Cited by 1 | Viewed by 1235
Abstract
With the reclassification of COVID-19 as an endemic disease and the relaxation of measures, Slovenia needed a complementary system for monitoring SARS-CoV-2 infections. This article provides an overview of the epidemiological situation of SARS-CoV-2 in Slovenia using a wastewater surveillance system, demonstrating its [...] Read more.
With the reclassification of COVID-19 as an endemic disease and the relaxation of measures, Slovenia needed a complementary system for monitoring SARS-CoV-2 infections. This article provides an overview of the epidemiological situation of SARS-CoV-2 in Slovenia using a wastewater surveillance system, demonstrating its usefulness as a complementary tool in epidemiological surveillance. This study found that estimated SARS-CoV-2 infections in Slovenia peaked in September 2022 and showed a declining trend with subsequent lower peaks in March–April and December 2023, mirroring the trends observed from clinical data. Based on both surveillance systems, the most prevalent variant in 2022 was BA.5. By 2023, BQ.1 and other Omicron variants increased in prevalence. By the end of 2023, XBB sublineages and the BA.2.86 variant had become predominant, demonstrating consistent dynamic shifts in variant distribution across both monitoring methods. This study found that wastewater surveillance at wastewater treatment plants in Slovenia effectively tracked SARS-CoV-2 infection trends, showing a moderate to strong correlation with clinical data and providing early indications of changes in infection trends and variant emergence. Despite limitations during periods of low virus concentration, the system proved significant in providing early warnings of infection trends and variant emergence, thus enhancing public health response capabilities. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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16 pages, 346 KiB  
Perspective
A Perspective on Wastewater and Environmental Surveillance as a Public Health Tool for Low- and Middle-Income Countries
by Mohammad Shehryaar Khan, Christian Wurzbacher, Anna Uchaikina, Boris Pleshkov, Olga Mirshina and Jörg E. Drewes
Microorganisms 2025, 13(2), 238; https://doi.org/10.3390/microorganisms13020238 - 22 Jan 2025
Viewed by 1537
Abstract
Geographical variations in infectious diseases create differences in public health priorities between high- and low-income countries. Low- and middle-income countries (LMICs) face resource constraints that limit adherence to international monitoring standards for wastewater-based epidemiology (WBE). The development of low-cost WBE programs, such as [...] Read more.
Geographical variations in infectious diseases create differences in public health priorities between high- and low-income countries. Low- and middle-income countries (LMICs) face resource constraints that limit adherence to international monitoring standards for wastewater-based epidemiology (WBE). The development of low-cost WBE programs, such as those to detect SARS-CoV-2, offers LMICs a promising tool for monitoring pathogens of local concern. In this work, we summarize important wastewater biomarkers for LMICs and their associated public health challenges, ranging from pathogens causing gastroenteritis to putative markers for plant diseases linked to food safety, as well as antimicrobial resistance. We raise awareness of the great potential of WBE for LMICs and highlight the critical health markers, research needs, and strategies necessary to establish tailored wastewater surveillance programs. Full article
(This article belongs to the Special Issue Surveillance of SARS-CoV-2 Employing Wastewater)
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