A scoping review of human pathogens detected in untreated human wastewater and sludge Open Access

The recent coronavirus disease 2019 (COVID-19) pandemic demonstrated that the risk of the emergence and rapid spread of new human health threats can only be combatted by innovative public health tools including strengthened surveillance networks and the integration of rapidly evolving tools, such as wastewater monitoring, into traditional surveillance systems (Mao et al. 2020; Baker et al. 2022).

Wastewater monitoring or surveillance is an approach to identify the presence or abundance of pathogens, biological or chemical indicators of disease, drug and substance use patterns, and other signals within a population over time and may inform public health decision-making (Mao et al. 2020; Sims & Kasprzyk-Hordern 2020). For the surveillance of human diseases, a wastewater monitoring approach can provide a less biased and cost-effective measure of disease prevalence as wastewater data are not dependent on human behavior and health system capacity (Safford et al. 2022; Kilaru et al. 2023). Wastewater monitoring has historically been used to identify pathogens transmitted through water and fecal-oral routes (Kilaru et al. 2023). However, wastewater monitoring has gained momentum globally with innovative research and developments in surveillance for respiratory pathogens during the COVID-19 pandemic (Tlhagale et al. 2022; Boehm et al. 2023). The potential to use wastewater to monitor a wide range of pathogens remains unclear (Kilaru et al. 2023). Therefore, the aim of this scoping review (ScR) was to identify and characterize the current evidence on human pathogens and antimicrobial resistance detected in untreated human wastewater and sludge.

An ScR uses systematic methodology to identify and map relevant evidence on an existing or emerging topic (Arksey & O'Malley 2005; Levac et al. 2010; Tricco et al. 2016; Peters et al. 2020a). While similar in the structure and rigor of systematic reviews, their purpose is to identify and map the landscape of the literature and determine possible gaps in the literature (Arksey & O'Malley 2005; Levac et al. 2010; Tricco et al. 2016; Peters et al. 2020a).

To ensure transparency, reproducibility, and consistency during all stages of the ScR, a protocol was created prior to conducting the ScR (Supplementary 1). The protocol includes a list of definitions, inclusion/exclusion criteria, search strategy, title and abstract screening forms, and the data characterization form. A few small deviations were made to the original protocol and are outlined in Supplementary 1. This ScR adheres to the Joanna Briggs Institute methodology for ScRs (Peters et al. 2020b) and was prepared in accordance with the PRISMA extension for ScRs (PRISMA-ScR) (Tricco et al. 2018). A multidisciplinary team with expertise in evidence synthesis, epidemiology, infectious diseases, wastewater, and public health created the protocol and executed the ScR.

The objective of this ScR was to identify and summarize relevant evidence that addresses the research question: what human pathogen(s) or antimicrobial resistant organisms/antimicrobial resistance genes have been detected through wastewater testing or surveillance of untreated, human wastewater or sludge? The following inclusion criteria were applied:

• (1)

  Publication date: All

• (2)

  Language: English and French

• (3)

  Study design: All

• (4)

  Country: All

• (5)

  Document type: Primary research (represents a study where the authors collected and analyzed their own data)

• (6)

  Pathogens: Human (known to infect and cause diseases in humans). Indicator organisms (bacteria and viruses that are used as surrogates to evaluate the presence of pathogens in water and to monitor water quality) were excluded, except if the study was looking at the presence of antimicrobial resistance genes.

• (7)

  Type of wastewater: Raw/untreated human wastewater or sludge

A comprehensive search strategy was developed by a Health Canada research librarian in collaboration with the authors and applied in five bibliographic databases: Scopus, Embase, Medline, Global Health, and Europe PMC (Supplementary 1). The initial search was conducted on February 15–17, 2022 and was updated on March 21, 2023.

The reference lists of relevant reviews on the topic were hand-searched to the point of saturation to ensure that the database search captured all relevant primary research. During the search verification process of 18 reviews, an additional 304 articles were identified and added to the search results (Hovi et al. 2012; O'Brien & Xagoraraki 2019; Bhatt et al. 2020; Corpuz et al. 2020; Hassoun-Kheir et al. 2020; Michael-Kordatou et al. 2020; Ali et al. 2021; Mousazadeh et al. 2021; O'Keeffe 2021; Pruden et al. 2021; Saawarn & Hait 2021; Zaatout et al. 2021; Bonanno Ferraro et al. 2022; Chau et al. 2022; Dzinamarira et al. 2022; Huang et al. 2022; Shah et al. 2022; Kilaru et al. 2023). Due to the volume of literature on the topic identified through the database search and a lack of resources, a grey literature search was not conducted.

Search results were imported into the web-based systematic review software DistillerSR (Version 2023.6.2; Evidence Partners, 2023) and duplicates were removed. If an included preprint was subsequently published after our search date, then the preprint version was manually replaced with the published version in DistillerSR, and extractions were completed on the published version. All stages of the ScR were conducted within DistillerSR, including relevance screening and data characterization.

A relevance screening form was developed to determine if the article was relevant to the review using the inclusion and exclusion criteria. The full text of potentially relevant articles was then procured and reviewed using a data characterization form, which aimed to confirm article relevance and extract data on pertinent information from each study such as study design, wastewater sample, sampling frequency and location, method of detection, and pathogens identified. For antimicrobial resistance, we captured whether the article studied antimicrobial resistant organisms in wastewater and how it was tested. Both forms (50 relevance screening and 10 data characterization) were developed a priori and piloted by all reviewers for clarity and consistency. Modifications were implemented based on reviewer feedback, where necessary, prior to implementation. Two reviewers independently used these forms to screen for relevance and extract data. During both stages, reviewers resolved conflicts by consensus or a third reviewer. Both forms including all definitions and data extracted are outlined in detail in the protocol (Supplementary 1). Excluded studies and the reasons for exclusion are documented in Supplementary 2.

The completed dataset was exported into Microsoft Excel^® for Microsoft 365 MSO (Version 2307 Build 16.0.16626.20198) for data cleaning, categorization, descriptive analysis, and narrative summarization. The complete dataset is available in Supplementary 3.

Articles were published between 1929 and 2023 with 56.5% (972/1,722) published since 2020. The majority of the articles were primary peer-reviewed research articles (1,525/1,722) and originated from Europe (569/1,722), North America (480/1,722), and Asia (413/1,722) (Table 1). The articles utilized observational (1,468/1,722) or experimental (580/1,722) study designs to investigate human pathogens in wastewater, with the majority being longitudinal/surveillance (includes monitoring) study designs (1,299/1,722). Experimental studies included the evaluation of detection methods (277/1,722), evaluation of sampling/sample preparation methods (254/1,722), or controlled or challenge trials (49/1,722), in which the majority analyzed the fate of pathogens through wastewater treatment plants. A small number of relevant primary research also included a risk assessment or predictive model (103/1,722). Many papers encompassed multiple study designs, most often to validate the detection of a pathogen in wastewater under controlled conditions prior to conducting a field study. The pathogen or antimicrobial resistance organism concentration or signal strength was quantified in 61.6% (1,060/1,722) of the studies. Untreated/raw wastewater was sampled in 97.3% (1,675/1,722) of studies, and untreated/raw/primary sludge was sampled in 5.9% (102/1,722) of studies.

The wastewater sampling details of the 1,722 included studies can be found in Table 2 and are described briefly below. The sampling of wastewater occurred in multiple locations including urban communities (1,350/1,722), small rural communities (<10,000 people, 85/1,722), buildings or facilities (372/1,722), and airplane/ship/other transport vehicles (12/1,722). Of the building or facility sample locations, the majority were hospitals (225/372) and universities/colleges (88/372), followed by residential buildings (20/372), schools/daycares/nurseries (18/372), retirement/nursing homes (17/372), retail/shopping (14/372), workplace/offices/factories (13/371), airports/bus terminals (11/372), quarantine/isolation centers (10/372), worker accommodations (9/372), prisons (6/372), hotels/hostels (4/372), and homeless shelters (4/372). For the sampling point, 73.2% (1,261/1,722) of studies obtain their samples from a wastewater treatment plant. Other sampling points included facilities/institutes (372/1,722), sewers (215/1,722), pumping stations (67/1,722), and open drains/canals (66/1,722). Composite (681/1,722), grab (470/1,722), and passive (72/1,722) sampling were the main methods used to collect wastewater samples. The sampling strategies were longitudinal sampling (1,440/1,722) or one-time detection (235/1,722). The most commonly reported sampling frequencies were weekly (278/1,722), one-time (265/1,722), and monthly (222/1,722). The study period was most often 1–2 years (368/1,722) followed by 1–3 months (360/1,722). Polymerase chain reaction (PCR) (1,085/1,722) and culture/biochemical identification (500/1,722) were the most frequently used methods of initially detecting pathogens in wastewater. PCR was also the most commonly used secondary or confirmatory detection method (435/1,722), followed by gel electrophoresis (325/1,722) and Sanger sequencing (326/1,722).

The detection of human pathogens in untreated wastewater or sludge was described in 99.3% (1,709/1,722) of studies. Of these, pathogens were reported at the genus or species level (1,655/1,709), family level (85/1,709), and at a higher taxonomic rank (14/1,709). Across these studies, 551 pathogens were described at the genus or species level, belonging to 142 distinct microbial families. There were also 10 additional microbial families studied without further taxonomic ranking being provided. A full list of pathogens found in wastewater is available in Supplementary 4.

Studies conducted prior to 2019, the year COVID-19 emerged, focused on pathogen families transmitted primarily through fecal–oral or waterborne pathways such as Enterobacteriaceae, Picornaviridae, and Caliciviridae, as shown in Figure 3. Starting in the early 2000s, research studies started exploring the detection of pathogens that were primarily transmitted through other pathways such as respiratory, vector-borne, sexual, and blood-borne. Aside from SARS-CoV-2 (n = 675), the number of research studies investigating other respiratory pathogens was low and included SARS-CoV-1 (n = 5) and MERS-CoV (n = 1), influenza A & B (n = 22), respiratory syncytial virus (RSV) (n = 9), and measles virus (n = 5). These studies were primarily conducted in 2020–2023, with the exception of one study on influenza A conducted in 2011 and three studies on SARS-CoV-1 conducted in 2005. Over the last 3 years, sporadic publications on vector-borne pathogens in wastewater have been published on chikungunya (n = 1), dengue (n = 2), yellow fever (n = 1), zika (n = 1), tularensis (n = 1), and tick-borne relapsing fever (n = 1). Starting in the late 2000s, several pathogens causing sexually transmitted and blood-borne infections have also been investigated, including herpes simplex viruses (n = 4), Neisseria gonorrhoeae (n = 1), Treponema pallidum (n = 1), and human papillomaviruses (HPV) (n = 13).

There were 6.4% (111/1,722) of studies where pathogens being studied were not detected in wastewater or sludge. This included 120 pathogens belonging to 65 distinct microbial families (Supplementary 4). Of these pathogens, 81.1% (99/122) were detected in wastewater or sludge in other studies and 18.9% (23/122) of pathogens were not detected in individual studies when they were looked for, including hantaviruses, Toxoplasma gondii, and simian immunodeficiency virus (SIV).

Wastewater detection of antimicrobial resistant organisms or genes was described in 20.6% (355/1,722) of studies. The majority of these studies were conducted in Europe (148/355) and Asia (119/355), followed by North America (45/355), Africa (34/355), Australia/Oceania (34/355), and South America (20/355). Antimicrobial susceptibility was tested phenotypically (109/355), genotypically (96/355), or using both methods (150/355). Escherichia coli (177/355) and Klebsiella pneumoniae (86/355) were the most commonly reported organisms in antimicrobial resistance studies.

Researchers connected their wastewater findings with population health data in 43.0% (741/1,722) of studies. The majority of these studies correlated wastewater results with clinical surveillance or case data (710/741) for SARS-CoV-2 (471/710), Enteroviruses (63/710), antimicrobial resistant organisms (55/710), or viruses causing acute gastroenteritis (rotaviruses, noroviruses, adenoviruses, sapoviruses, and astroviruses) (49/710).

Wastewater detection or monitoring of pathogens was reported to lead to public health action in 3.4% (58/1,722) of studies. This included decisions to commence (43/58) or cease (1/58) public health measures (e.g., additional surveillance, targeted testing, and isolation), implement public health messaging (8/58), and support decision-making (8/58). The majority of these studies were on SARS-CoV-2 (43/58) followed by poliovirus (7/58).

This ScR summarizes the characteristics of the global research on human pathogens and antimicrobial resistance targets found in untreated wastewater and sludge. The results demonstrate that a wide range of pathogens can be detected in wastewater. However, the usefulness of wastewater monitoring for different pathogens is dependent on both the burden of disease in a community, as well as if and how much the pathogen is excreted into wastewater during infection.

In this ScR, there are several notable topic areas that are not underpinned by many studies including certain pathogens, antimicrobial resistance, and some methods for wastewater monitoring. Most studies focused on viral and bacterial pathogens with minimal research on other pathogen types, protozoa, helminths and fungi; the latter are a growing international concern (Benedict et al. 2017; Nnadi & Carter 2021). Some priority fungal pathogens designated by the World Health Organization have been identified in wastewater studies including Candida auris and Nakaseomyces glabratus (Brumfield et al. 2022; World Health Organization 2022; Barber et al. 2023; Rossi et al. 2023). However, this ScR shows that more research is needed to determine if wastewater monitoring is an appropriate method for identifying emerging fungi of international concern. Further syntheses on the identified studies related to antimicrobial resistance may be warranted to understand how and why wastewater has been used to address this important global public health threat.

Tracking antimicrobial resistance burden and trends is an area for which wastewater monitoring could potentially be useful; however, this ScR identified little research on strategies or utility for such monitoring. In theory, monitoring antimicrobial resistance in wastewater could be beneficial for the purpose of identifying multidrug resistant pathogens currently circulating at the population level. Indeed, this is data that has historically been difficult to acquire, and it may provide insights into potential resistance acquirement and dissemination in the environment as sewage can contain a confluence of antimicrobials, human pathogens, resistance genes, and mobile genetic elements (Liguori et al. 2022).

Within the current literature, most of the studies on sampling, sample handling, and extrapolation of wastewater results to the burden of infection in a population were conducted on SARS-CoV-2. For other pathogens, there were few studies that examined the sensitivity of methodologies used to understand the emergence or fluctuations of a pathogen within a population. As the utility of wastewater monitoring is being considered for a broader range of pathogens, research to establish the best practices will need to be conducted. Wastewater samples were the most common for monitoring human pathogens compared to sludge samples. In studies examining both, sludge samples have been described as containing a broader diversity of human pathogens but may also have more inhibitors and other contaminants that change the sample handling and extrapolation of results. Thus, more research is needed into the assay sensitivity, sample processing, and result extrapolation trade-offs for different sample types and how this may influence pathogen detection within a community wastewater catchment (Bibby & Peccia 2013; Peccia et al. 2020). Optimal sampling frequencies may vary depending on the pathogen and characteristics of the outbreak or circulation of the pathogen in the population. Studies conducted on SARS-CoV-2 show that differences in sampling frequencies influenced the characterization of incident COVID-19 cases at the population level (Chan et al. 2023). Daily data were described as the ‘gold standard’ for the trend analysis of SARS-CoV-2 and a minimum of 2–5 samples per week were necessary for capturing trends within the community for SARS-CoV-2 (Chan et al. 2023; Kuroita et al. 2023). For other pathogens, daily and twice weekly sampling approaches are less common in wastewater monitoring, and most studies reported weekly or monthly sampling. An evaluation of the optimal sampling strategy to capture community trends would need to be established for each pathogen and may vary depending on the epidemiologic situation (e.g., outbreak vs. seasonal activity) and the goals of the monitoring system.

This ScR identified that a limited number of studies were conducted in the Caribbean, Central America, Africa, and the Middle East. Wastewater monitoring may offer the greatest benefit in countries with limited clinical surveillance capacity, especially considering that wastewater has several advantages over sampling individuals and case counting, including its ability to be a cost-effective strategy for the detection and evaluation of the burden of infectious diseases in a population (Ali et al. 2022; Ngwira et al. 2022). Considerations for the application of wastewater monitoring in developing countries include an assessment of the wastewater infrastructure and the design of a sampling strategy to get representative samples (Pandey et al. 2021).

It is our intention that the information synthesized and knowledge gaps discussed in this ScR will be used to support evidence-informed decision-making and further research on this topic. Although we attempted to capture all relevant literature on this topic, it is possible that some research was not captured due to the complexity of the search, publication bias, absence of citation indexing, lack of citations by included papers, or the omission of an extensive grey literature search. Articles published in any language other than English and French were not characterized in this ScR due to translation resource constraints; however, only two studies were excluded on the basis of publication language.

While wastewater testing to monitor pathogens is not a new concept, the success of using wastewater to detect SARS-CoV-2 and estimate community burden during the pandemic has demonstrated the use of wastewater to inform public health responses and provided a unique opportunity to compare the traditional surveillance methods against wastewater monitoring data for a sustained period of time. Outside of SARS-CoV-2, a few studies on enteric pathogens also correlated wastewater signals to clinical surveillance data. These suggest that a multifaceted approach to surveillance that incorporates wastewater signals and other traditional epidemiological metrics (e.g., incidence, hospitalizations, and pharmaceutical sales) would be beneficial for both outbreak situations and routine monitoring of many pathogens, including but not limited to influenza, RSV, and antimicrobial resistance. Recent research on SARS-CoV-2 and mpox has shown that wastewater can be used to monitor the burden and spread of a pathogen in a population fairly quickly and economically, which has the potential to influence case-based surveillance strategies in the future (Sherchan et al. 2023; Tiwari et al. 2023). From a public health preparedness lens, additional research is needed to determine which pathogens are conducive to wastewater monitoring, and where, when, and how monitoring should be implemented.

The authors acknowledge Iryna Artyukh, Adhiba Nilormi, Caroline Larocque Guzman, Melanie Katz, Sydney Jennings, Dima Ayache, and Mavra Qamar for assisting with relevance screening and data characterization. Also, David Champredon and Manon Fleury for their expertise and input into the conceptualization of this project, Natalie Knox and Anil Nichani for their project support and expertise, and the Health Canada library for help in designing the search strategy and article procurement.

T.C. served as the project lead, contributed to the design, performed as a reviewer, and wrote the manuscript. P.R. was the project lead, contributed to the design, performed as a reviewer, and edited the manuscript. K.Y. contributed to the design and manuscript, performed as a reviewer, and edited the manuscript. G.M. served as a reviewer, contributed to and edited the manuscript. A.B. performed as a reviewer and data cleanup, and edited the manuscript. K.P. performed as a reviewer and edited the manuscript. L.W. was responsible for the conceptualization, design and initiation of the project, and also edited the manuscript, supervisor. All authors approved the final version for publication.

All relevant data are included in the paper or its Supplementary Information (Supplementary Files 1–4: https://doi.org/10.17605/OSF.IO/2Y6ZU).

The authors declare there is no conflict.