https://orcid.org/0000-0001-9611-785X
Figures
Abstract
In response to COVID-19, wastewater surveillance rapidly expanded in high-income sewered settings. However, little is known about sampling in at-risk areas without sewerage infrastructure. We developed a method to test waste effluent streams, including: identifying environmental virus detection and waste effluent locations for testing; determining appropriate sampling methods; and, safely collecting and analyzing samples. In Côte d’Ivoire, we identified waste canals from urban slum areas and liquid waste streams in chicken slaughtering markets as high risk for SARS-CoV-2 and Influenza A Virus (IAV), respectively. For 12 weeks, we sampled once per week for SARS-CoV-2 using active and passive sampling at two canals downstream of urban slum areas containing primarily human wastewater; and IAV using passive sampling at one site containing animal slaughtering wastes (in duplicate). Samples were prepared, extracted, and processed using RT-PCR in Côte d’Ivoire. Of 48 SARS-CoV-2 samples, 22 (13 active, 9 passive) tested positive (Ct values 28.2-35.1). Of 24 IAV passive samples, three (12.5%) tested positive (Ct values 29.3-32.4). We successfully used our methods to identify relevant viral pathogens with diverse host ranges and provide proof-of-concept for sampling priority pathogens in waste effluent streams. Our work provides a pathway to democratize and extend gains of wastewater surveillance to at-risk populations in non-sewered contexts without wastewater infrastructure. Further research is needed to develop waste effluent surveillance in non-sewered settings along the entire chain of understanding high-risk viral pathogens in waste effluent, including: laboratory research to understand pathogen survivability in various media; understanding where in the environment high-risk pathogens might be; determining how to sample and test various waste effluent streams; and, in establishing the collaborative partnerships and training to complete high-quality research and expand these results to additional pathogens.
Citation: Yapi EAM, Mossoun AM, Zan-Bi TT, Dindé AO, Gossé LG, Vakou SN, et al. (2025) Expanding access to wastewater surveillance beyond sewered networks: Effectiveness of active and passive sampling of waste effluent streams in Côte d’Ivoire. PLOS Water 4(6): e0000290. https://doi.org/10.1371/journal.pwat.0000290
Editor: Vikram Kapoor, University of Texas at San Antonio, UNITED STATES OF AMERICA
Received: July 29, 2024; Accepted: May 28, 2025; Published: June 27, 2025
Copyright: © 2025 Yapi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are in the manuscript.
Funding: This publication was made possible through the support provided to Tufts University by the Emerging Threats Division, Office of Infectious Disease, Bureau for Global Health, U.S. Agency for International Development. The opinions expressed herein are those of the authors and do not necessarily reflect the views of the U.S. Agency for International Development. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The COVID-19 pandemic led to a rapid expansion of wastewater surveillance to establish population-level disease trends [1]. While wastewater surveillance does not provide information about individual disease burden, wastewater surveillance can be used to predict population-level near-future disease burden, understand trends for diseases that may have delayed or unavailable data from clinical sources, and can be used in conjunction with epidemiological data to estimate or model the current population burden of disease [2–5]. These uses are powerful, and community-level pooled surveillance can be used as tool to deploy public health response efforts and other resources to areas with demonstrated, anticipated, or predicted increases in disease burden.
In areas with sanitary or combined sewerage systems, feces, urine, and wash water flow from toilets, showers, and sinks through pipes in the home to sewerage pipes under the ground to a centralized wastewater treatment facility [1]. To conduct wastewater surveillance, a sample is collected from the sewer network, most often from a central point with large catchment populations (such as near the wastewater treatment plant intake pipe). Commonly, multiple samples are collected over time for each location in an automated sampler and pooled for testing. In high-income contexts with sewerage systems, wastewater surveillance is now used for a greater and continually expanding range of pathogens beyond SARS-CoV-2, including influenza viruses, RSV, mpox, and dengue [6–10]..
However, the gains of wastewater surveillance have largely not, to date, been realized in areas without sewer networks. This is due to challenges associated with sampling from, and testing, decentralized waste sources [11]. The Joint Monitoring Programme (JMP) of UNICEF/World Health Organization measures progress toward the Sustainable Development Goals for water, sanitation, and hygiene [12]. The JMP has established “ladders” to benchmark and compare service levels. For sanitation, JMP considers “safely managed sanitation service” the highest tier of the sanitation service ladder. A safely managed service is when people: 1) use improved sanitation facilities (designed to hygienically separate excreta from human contact); 2) do not share that service with other households; and, 3) the excreta is treated and disposed of in-situ; stored, emptied, and treated off-site; or, transported via a sewer network and treated off-site. Lower tiers of the sanitation ladder include people using improved sanitation facilities that are not safely managed, using shared facilities, using unimproved facilities (such as a pit latrine) or practicing open defecation. Currently, while the vast majority of wastewater surveillance is conducted in populations with safely managed sanitation [13], the JMP estimates that, in 2022, 3.5 billion people (nearly half of the global population) still lack safely managed sanitation. These same populations also have weaker clinical monitoring of disease, exacerbating difficulties in understanding the disease burden and disease trends.
There is a need to extend the benefits of wastewater surveillance to areas without safely managed sanitation. This requires development of sampling frameworks, sampling methods, and analysis methods. One promising sampling method to extend benefits of wastewater surveillance, is the use of passive samplers. Passive sampling can be used to capture wastewater in absorbent material to produce a composited sample without the use of expensive equipment or much additional resources [14,15].
The objective of our work presented herein was to extend the benefits of wastewater surveillance to non-sewered areas at risk of spillover in Côte d’Ivoire, using novel frameworks and methods.
Materials and methods
To complete our objective of testing low-prevalence spillover-risk pathogens in the environment, the authors worked collaboratively to: 1) review the research and local knowledge to identify high-risk waste effluent sampling locations; 2) select appropriate sampling methods for the media in those exact locations; 3) safely collect and transport samples to the laboratory; and, 4) analyze samples with appropriate testing methods (Fig 1). Each of these are described below.
Selection of sampling locations
The project team met, reviewed data, visited sites, and selected two locations in Côte d’Ivoire for initial sampling: 1) open small canals that collect human wastewater from slums in Yopougon (Abidjan); and, 2) open small waste streams that collect chicken slaughtering liquid waste at a poultry market in Grand Bassam. These discussions included two laboratories in Côte d’Ivoire, L’Institut Pasteur de Côte d’Ivoire (IPCI) and Le Laboratoire National d’Appui au Développement Agricole (LANADA), who, had experience sampling SARS-CoV-2 and IAV [16], respectively. The laboratories’ previous experience in testing biological samples for these pathogens was a key reason for selecting these pathogens in this initial proof-of-concept trial.
Sample collection
All sample collection and analysis procedures followed approved biorisk management practices appropriate for the working environment and risks. This work was determined to be non-human subjects research by the Tufts University Institutional Review Board and appropriate authorities in Côte d’Ivoire.
In the morning, trained laboratory staff arrived at the waste canal with the materials necessary to safely place the passive sampler in the waste stream and also collect an active sample (also known as a grab sample) to test for SARS-CoV-2. Staff donned appropriate personal protective equipment (PPE) (e.g., gloves, masks, gowns) before placing the passive sampler (Live Better or Organyc® brand 100% cotton organic tampon) in the waste stream by tying the string of the sampler to a rope, wrapping the rope around a rock to anchor the sampler, and securely attaching the end of the rope around an iron bar embedded in the ground above the waste canal. The sampler was left in place for 4–6 hours, with a sign saying not to disturb. An active sample was also collected at this time, by lowering a clean bucket on a rope into the canal, collected water, and pouring it directly into a labelled, clean 500 mL high-density polyethylene (HDPE) plastic bottle; which was put on ice and transported to the lab within two hours. Following all active and passive sampling at both sites, staff safely doffed and placed materials (including used PPE) in a biohazard bag for disposal at the laboratory.
In the morning, trained laboratory staff arrived at the poultry market with the materials necessary to safely place the passive sampler in the waste stream to test IAV. Staff donned appropriate PPE (as described above) before placing two passive samplers (duplicates) in the waste stream by tying the string of the sampler to a rope, and securely attaching the end of the rope around a bench. Staff safely doffed and placed materials (including used PPE) in a biohazard bag for disposal at the laboratory. The samplers were left in place for 4–6 hours, with a sign saying not to disturb.
In the afternoon, trained laboratory staff arrived at both locations with the materials necessary to safely collect the passive samplers. Staff donned appropriate safety equipment before carefully raising the sampler from the waste effluent stream and placing it into a Whirl-Pak™ bag (Chicago, IL, USA). The bag was closed by wrapping (not whirling), disinfected with chlorine solution, placed in a labelled secondary plastic container with absorbent material on ice, and transported to the laboratory for processing within two hours.
Upon arrival at the laboratories, all samples were pasteurized in a water bath inside the HDPE container. For passive samples, each sampler was transferred to a biosafety cabinet and manually squeezed for two minutes from outside of the Whirl-Pak™ bag, followed by cutting the bottom corner of the bag and transferring liquid and passive sampler into a syringe with 20 mL of phosphate buffed saline (PBS) and Tween20 solutions (10 mM sodium phosphate, 0.15 M NaCl; 0.05% Tween20) for SARS-CoV-2, and 20 mL of PBS solution for IAV. The syringe was used to extract the liquid into tubes. For active sampling, waste effluent was directly and carefully poured into tubes. All capped tubes were centrifuged at ~400xg for 10 minutes at 4°C to precipitate coarse debris, and then 15 mL of supernatant was transferred to an Amicon Ultra-15 ultrafiltration unit (Merck, Darmstadt, Germany). Amicon tubes were centrifuged at ~2,700xg for 35 minutes at 4C. Centrifugation was repeated until retentate volume was 140–400 μL. Then, 140 μL of retentate was transferred to a 1.5 mL RNAse-free microcentrifuge tube for RNA extraction.
RNA extraction was immediately conducted using the Qiagen AllPrep PowerViral wastewater kit for RNA extraction (Venlo, The Netherlands) for SARS-CoV-2, and the QIAmp Viral RNA kit for IAV (Venlo, The Netherlands). Both kits were used according to manufacturer’s instructions. RNA was detected and quantified by RT-qPCR. For SARS-CoV-2, a SD Biosensor kit and primers and probes at the laboratory were used for detection; for IAV primers and probes were used.
Data analysis
Data were entered into Microsoft Excel (Redmond, WA, USA), and included sample ID, date and location of sampling, Cycle threshold (Ct) value, and positive/negative determination. Sample PCR results are presented in number and percent positive, and Ct values are presented in mean (stdev, range). Passive and active sample SARS-CoV-2 results were compared using Fisher’s Exact Test in StataSE (College Station, TX, USA), and passive and active SARS-CoV-2 Ct values were compared using a two-tailed non-paired t-test in Excel.
Results
In July 2023, Tufts University personnel visited potential sampling sites in Côte d’Ivoire, trained laboratory staff on sample collection, and developed the project standard operating procedures (SOPs). During this visit, it was determined that we would sample for SARS-CoV-2 (at two waste canals) using both active and passive sampling; and, at a poultry market using active sampling only (Fig 2).
Over 12 weeks from January to March 2024, the project team collected 48 SARS-CoV-2 samples. These included 12 weeks of sampling from two waste streams, for a total of 24 passive and 24 active SARS-CoV-2 samples. IAV passive samples were collected in duplicate (12 weeks, duplicates) for a total of 24 samples.
Of the 48 SARS-CoV-2 samples, 22 (46%) tested positive; including 13 of 24 active samples (54%) and 9 of 24 passive samples (38%) (Fig 3). Results from the two sites were pooled as sites were near to one another. These results were not significantly different by Fischer’s Exact Test (p = 0.193). The average Ct value for active samples was 30.8 (range 28.2-33.5, stdev 1.4), and for passive samples was 31.6 (range 28.8-35.1, stdev 1.8). These values were not significantly different by T-test (p = 0.193). Overall, the low Ct values indicate a low but detectable quantity of SARS-CoV-2 RNA in these samples, and these Ct values are in line with the lowest amounts detected in wastewater samples from sewered systems. While there were non-significant differences between active and passive sampling, both were able to detect evidence of this common viral pathogen in waste effluent streams.
IAV samples were collected in duplicate (at the same location on the same day) for 12 weeks (24 samples total) (Fig 4). Of these 24 IAV samples, 3 (12.5%) tested positive. During Week 2, duplicates tested positive, with Ct values of 29.3 and 29.5. In Week 10, the Ct value was higher, at 32.4, and only one of the two duplicates tested positive.
Discussion
The successes of this research include a large international collaboration collaborating to show proof-of-concept for passive sampling of spillover-risk human and animal pathogens in waste effluent streams in Côte d’Ivoire. This demonstrates a low-cost and highly effective technique for performing community-level disease surveillance in regions without piped sewage networks, using local in-country government-affiliated laboratories. Our methods can continue to be developed to democratize access to wastewater sampling. Additionally, weekly reports were provided to government ministries in Côte d’Ivoire, and USAID. There was significant interest by those agencies in the potential for expansion of this work.
This work was not without limitation and challenges. The main limitation was a lack of detail in reported laboratory data (e.g., standard curves, controls that can be used to calculate false positive and false negative rates) commonly seen in wastewater sampling in high-resource contexts. As donor and government policies support localization of laboratory testing, this project was required to work with local government approved laboratories to complete all testing. Additionally, all analysis was required to be completed in country. This led to the successful collaboration described in the previous paragraph. However, it also led to receiving results in local reporting formats, which do not provide all the details provided or expected in high-income country laboratory reporting. We acknowledge this as a limitation of our results in this manuscript. The results presented herein should be understood within this framework. Please note that this limitation does not impact the novel methods we have developed to assess low-prevalence spillover risk from waste effluent streams in low-resource settings, or the proof-of-concept of those methods. Please also note there the methods presented herein could also be utilized in high-income settings, to assess low-prevalence pathogen spillover risk.
There was also a lack of ability to sequence results in country, and as such sequencing was not completed. Additional challenges of this work included that laboratory supplies and PPE were imported to Côte d’Ivoire, and more training and supplies would be needed for additional PCR results, sequencing, and expansion to higher risk (spillover) pathogens. To address these limitations and challenges in the future, it is recommended to provide equipment, supplies, and training to in-country, government-supported laboratories. In fact, currently, IPCI and LANADA are receiving facility upgrades to enhance their high-risk viral pathogen testing capacity, from World Health Organization and the Food and Agriculture Organization of the United Nations, respectively. In order to effectively make use of these facility upgrades, it is also necessary to invest in human resources, and provide the ongoing training and support to develop laboratory personnel with the experience to conduct testing and report results.
There is currently strong interest from donors and governments in wastewater-based epidemiology (WBE) for infectious disease surveillance. Previous work in African upper-middle-income countries has shown the benefits of WBE. For example, during the COVID-19 pandemic, South Africa implemented WBE across 87 wastewater treatment plants, successfully detecting SARS-CoV-2 RNA concentrations that correlated with clinical case data [17]. This approach provided a two- to three-week lead time in anticipating infection waves, demonstrating potential as an early warning system. Similarly, in Morocco, WBE was used to monitor SARS-CoV-2 and Influenza A, with findings aligning closely with reported cases, underscoring the method’s effectiveness in tracking respiratory pathogens [18]. Additionally, WBE has been proposed for monitoring neglected tropical diseases, which are often underreported due to limited healthcare access [19], and has been implemented successfully to identify multi-drug resistant tuberculosis [20]. Overall, WBE can inform public health responses in regions with constrained medical infrastructure.
Despite its advantages, the implementation of WBE in LMICs faces several challenges, particularly in low-income and lower-middle-income countries (such as Côte d’Ivoire). These limitations are documented in the literature and were seen in our works, such as: infrastructure limitations, such as limited centralized sewage systems hindering the collection of representative wastewater samples; and, lack of standardized protocols and limited laboratory capacity complicating analysis and interpretation of results [18].
To begin to address these limitations, World Health Organization (WHO) has recently issued draft pilot guidance on wastewater and environmental surveillance (WES) for infectious diseases, including in LMICs, that aims to provide “an overview framework for prioritization, implementation and integration of WES as part of multi-modal public health surveillance” [21]. Additionally, WHO has developed pathogen-specific summaries, including for SARS-CoV-2 [22] and Typhoid and Paratyphoid [23]. It is of note that these pathogen-specific guidance documents conclude “In non-sewered settings, based on limited evidence, WES for SARS-CoV-2 has moderate potential” and “there is inadequate evidence to determine the optimal contribution of WES to typhoid (and paratyphoid) disease surveillance and response”. WHO is supporting countries in implementing WES, and highlighting – as we saw in the work presented herein – that investments in infrastructure development, capacity building, and methodological standardization are essential.
Further research and next steps are needed to continue democratizing access to wastewater surveillance. The next steps of this project are sampling Lassa Virus in waste effluent of rural hospitals without reliable access to rapid test kits, to determine if Lassa Fever is prevalent yet undetected in Liberia and Côte d’Ivoire. We hope to also extend testing to additional pathogens on other project, such as cholera and Ebola in outbreak settings, including sequencing. Additionally, further research is needed along the entire chain of understanding high-risk viral pathogens in waste effluent: from laboratory research, to understand pathogen survivability in various media, to understanding where in the environment high-risk pathogens might be, to determining how to sample and test various waste effluent streams, to establishing the collaborative partnerships and training (including of local laboratory personnel) to complete this work.
Conclusions
In response to the COVID-19 pandemic, wastewater surveillance rapidly expanded in high-income settings. However, methods were not available to test wastewater in areas without sewerage infrastructure. We developed methods to test waste effluent streams in Côte d’Ivoire, and successfully used these methods to identify human- and animal-derived pathogens. Our work provides a pathway to democratize and extend gains of wastewater surveillance to at-risk populations with non-sewered waste effluent systems.
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