Wastewater and environmental surveillance for Vibrio cholerae: a scoping review Open Access

Cholera, a waterborne disease caused by the bacterium Vibrio cholerae, poses a significant public health challenge. It is often referred to as a disease of poverty due to its disproportionate impact on communities with limited resources, inadequate sanitation, and poor access to clean water (WHO 2023). There are approximately 1.3–4 million cholera cases annually and up to 143,000 deaths attributed to the disease (WHO 2023). Recent reports revealed that between 2022 and 2023, there was a 13 and 71% increase in cholera cases and deaths, respectively (WHO 2023). There are many V. cholerae serotypes but only two, namely O1 and O139, cause outbreaks (WHO 2023). Clinical manifestations of cholera include watery diarrhoea, vomiting, and dehydration, which can lead to severe complications and death if left untreated (WHO 2023). V. cholerae is mainly transmitted through the faecal–oral route (Baker-Austin et al. 2018). In the past few years, there has been a surge in cholera both in case numbers and geographical distribution (WHO 2023). In 2022 the WHO confirmed that the global risk of cholera remains at a critical level, posing a significant threat to public health. This highlights the persistence of cholera as a crucial indicator of social inequality and insufficient development (WHO 2022a, 2022b). Affected countries are encouraged to enhance disease surveillance and bolster national preparedness to swiftly identify and address outbreaks, employing a combination of surveillance, water, sanitation and hygiene, social mobilization, treatment, and oral cholera vaccines (WHO 2022a, 2022b).

The knowledge and experience gained from the application of WES for numerous diseases, including polio and COVID-19, underscores its significant potential contribution and added value to public health efforts (Berchenko et al. 2017; Ahmed et al. 2021). To date, there is a scarcity of comprehensive data regarding the effectiveness of WES in supporting outbreak response efforts for cholera. This scoping review aims to identify and describe the current evidence regarding the application of WES to act as an early warning system, to monitor trends in ongoing outbreaks, and to describe serogroups and virulent strains of toxigenic V. cholerae circulating in the community.

We conducted this scoping review using a pre-defined protocol in accordance with the Joanna Briggs Institute methodology for scoping reviews (Peters et al. 2022). The results of the scoping review were reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) checklist (Tricco et al. 2018). We did not critically appraise included studies, as this is not required for scoping reviews (Tricco et al. 2016).

We included field-based studies which involved fieldwork during which samples were collected in a natural setting of (1) wastewater (centralized/sewered or non-centralized/non-sewered), (2) wastewater-impacted environmental waters, and/or (3) drinking water sources (whether treated or untreated) to detect the presence of toxigenic V. cholerae without restriction on date or existence of an outbreak. We did not restrict studies according to sampling intervals and included once-off testing and periodic and continuous surveillance. We included original research articles of all designs (retrospective and prospective studies). We did not restrict studies according to geographic location, publication language, or date. We excluded studies that focused on non-pathogenic cholera genes (e.g., non-O1 or non-O139/ non-El Tor/classical), aquaculture, marine environments, exclusively clinical samples, non-field (laboratory-based) research and those that tested solely for faecal indicators (e.g., Escherichia coli).

We conducted a comprehensive and systematic literature search between May and June 2023 on PubMed and Scopus using a pre-defined search strategy (see the Supplementary Material 1). PubMed and Scopus provide comprehensive cover for sciences and are both accessible to search. Scopus is generally considered to be more comprehensive and has more citations than Web of Science. PubMed had more outputs, providing more eligible studies for the review. As this is a descriptive scoping review, rather than a systematic review of effectiveness, we opted to have a faster process, and not include more than two databases, which is the minimum number of electronic databases to adhere to quality standards for reviews. We ran an updated search in PubMed in January 2025, the database which found a greater number of records and added this detail to the publication (see the Supplementary Material 1). One author developed the search strategy using a combination of pre-defined keywords, with the assistance of a search specialist and input from the review team. We initially developed the search for PubMed and adapted it to the Scopus database. We uploaded the search results onto Covidence software (Kellermeyer et al. 2018) for deduplication and screening of studies. We further screened reference lists of relevant systematic or narrative reviews identified through the search to identify potentially eligible studies.

Authors independently and in duplicate screened the titles and abstracts of studies against the eligibility criteria and retrieved full texts of potentially relevant studies. We resolved discrepancies regarding the inclusion/exclusion of studies by consensus during author team meetings.

We developed and piloted a data extraction tool on Microsoft Excel (Washington, USA). Data extracted from relevant studies included title and year of publication, country of origin, study aims, type of sample, sampling technique, duration of sampling, frequency of sampling, volume of samples collected, number of samples, sample preparation methods, concentration method, and V. cholerae strain (see the Supplementary Material 2). We conducted independent duplicate extraction and compared the results to verify the data, resolving discrepancies with discussion with the author team.

We analysed data descriptively using Microsoft Excel. Pivot tables were created to organize and summarise the data extracted from included studies. These pivot tables were grouped thematically, based on various study characteristics captured through the extraction tool. Cross-tabulation within and across pivot tables was done to identify correlations in the data. The pivot tables were also used for data visualisation in the form of graphs and tables.

The studies were published between 1970 and 2024, with a peak in publications in 2018. However, upon examining the start and end year of sampling for these studies, no clear factors emerge to explain this peak. Notably, Torresi et al. (2018) collected samples between 2010–2011 and Bwire et al. (2018) undertook their sample collection between 2015 and 2016, yet both studies were published years later in 2018. This lag between fieldwork completion and publication limits the effectiveness of using the findings for disease management and further highlights the need for timely dissemination of research to enhance response efforts. Therefore, significant efforts are required to reduce publication delays and improve access to research findings, ultimately strengthening the evidence base on the presence of V. cholerae in the environment. Remarkably, 45% of the studies included in this review were published between 2010 and 2019. Since 2021, there has been a global rise in cholera cases and the spread across various regions (WHO 2022a, 2022b). It is hypothesized that the management of various outbreaks, particularly the ongoing COVID-19 pandemic, may have impacted prioritization and resources allocated to WES for cholera during this period.

Examining the geographical locations of these studies, it is evident that WES has predominantly been conducted in areas where cholera poses a significant public health threat. In most cases, cholera is endemic in these regions, with transmission exacerbated by inadequate water, sanitation, and hygiene (WASH) conditions. Most of the studies originated from lower-middle-income countries, underscoring the economic context of water quality issues in resource-limited settings. In contrast, a smaller fraction of studies came from upper-middle-income and high-income countries, with only 5% emerging from low-income countries. The number of studies conducted in low-income countries affirms the vulnerability faced by resource-constrained settings, characterized by poor water quality and health system infrastructure along with limited resources for locally relevant research (Global Task Force on Cholera Control 2022). This aligns with a recent systematic review that highlighted the global inequality in response to epidemics as gauged by the number of epidemics in low-income settings and the paucity of evaluation reports (Warsame et al. 2020).

Geographically, the South-East Asia continental region emerged as the most prominent location for research of this nature, accounting for 44% of studies. Historically, the origins of cholera strains have been traced back to South and South-East Asia, with six of the seven pandemics originating from the region (Lopez et al. 2020). Notably, the Eastern Mediterranean region (EMR) was the least studied, with 5% of studies conducted there. The EMR is a complex setting where the transmission and spread of cholera are driven by conflict, political instability, internal displacement of large segments of the population, as well as limited water and sanitation infrastructure (Buliva et al. 2023). In 2022, the EMR experienced a resurgence in cholera outbreaks across eight of its member states, with approximately 375 cholera-related deaths (WHO 2022a, 2022b). The Americas reported a considerable 27% of the studies, while Africa, Europe, and the Western Pacific regions were less frequently represented with 16, 11, and 9% of the studies conducted in these regions, respectively (Table 1). The number of studies reported from Africa may be indicative of resource constraints that have limited the capacity of countries to conduct WES.

The undertaking of WES of V. cholerae in Bangladesh is also reflective of the challenges the country faces in disease transmission. There are estimates that 100,000 cases and 4,500 deaths are reported each year in Bangladesh (Ali et al. 2015; Zaman et al. 2020). Moreover, cholera is endemic and seasonal in Bangladesh with transmission occurring consistently during the year (Das et al. 2023).

In comparison, in countries such as the United States, the occurrence of cholera outbreaks is rare. However, as evidenced in studies by Ceccarelli et al. (2015) and Daboul et al. (2020), there remains interest in the USA to investigate the presence of V. cholerae around coastal areas and estuaries in the country. With regards to study locations in the USA (n = 9), a higher proportion (44%) of these studies were conducted in Louisiana, reflecting regional research priorities, research interests, or environmental concerns. Notably in 2005, Louisiana was struck by devastating impacts of Hurricanes Katrina and Rita. However, no ‘direct flood-associated’ cholera cases or outbreaks were associated with these two extreme weather events (CDC 2006; Liang & Messenger 2018).

Low-income countries are also stifled by an overburdened healthcare system and a limited number of qualified personnel resulting in poor health research capacity. Two of the studies from Uganda reported that the processed samples were shipped to US-based laboratories for polymerase chain reaction (PCR) analysis. Although these studies were based on environmental monitoring of V. cholerae, transportation of samples to high-income countries may not be cost-effective and may lead to longer turnaround times during outbreaks (Elbireer et al. 2011). Investment in medical research laboratories with appropriate infrastructure would improve WES to monitor trends in ongoing outbreaks (Jayatilleke 2020).

In line with principles of Good Laboratory Practices, which encompass meticulous protocols for sample handling, transportation, and methods reporting, our review sheds light on the potential impacts of inadequate procedures (or reporting thereof) on sample integrity and data reliability. The transportation of samples, especially across long distances, can compromise sample integrity. According to WHO (2024), adherence to standard operating procedures for sample collection and transportation will minimise sample deterioration and ensure that samples arriving at the laboratory will provide good quality results. However, few studies gave details on the transportation conditions, particularly temperature management and controls. According to Ensor et al. (2022), weekly sampling is suitable to detect or confirm the presence of toxigenic V. cholerae, while sampling 2-3 times a week provides a better indication when monitoring toxigenic trends or outbreaks. Among the studies that detailed their sampling frequency (n = 70), 17% reported having collected samples on a monthly basis, whereas three studies utilized a multi-frequency strategy for sample collection. Therefore, to enhance the potential of WES to monitor trends in ongoing outbreaks, future studies need to consider more frequent sampling routines. In this review, there were no links between the sampling frequency and a country's income classification, or the types of samples collected.

Of the samples collected, a quarter of the studies concentrated on drinking water in their sampling strategy, with 79 and 79% of studies having collected wastewater-impacted environmental water, and wastewater samples, respectively (Table 1). The collection of wastewater-impacted environmental water samples in these studies reflects public health concerns around water bodies such as rivers, lakes, and ponds acting as reservoirs for V. cholerae. This is reflected in the work by Alam et al. (2015) in Haiti which assessed the role of surface water in the transmission of cholera and explored the extent to which V. cholerae has established environmental reservoirs in the country. Although only a quarter of studies focused on drinking water, surveillance of this type of water source can provide insight into the transmission routes for cholera within and between communities. In their investigation, George et al. (2017) found that drinking water and clinical isolates within households were more closely matched than isolates from outside households. This supported the findings that the source of the water-to-person transmission of cholera in the communities was indeed drinking water.

It is noteworthy that 66% of studies did not comment on the environmental conditions conducive to V. cholerae growth, which include water and ambient air temperature, rainfall, and salinity. The omission of environmental conditions in the occurrence and transmission of V. cholerae is surprising, given findings from studies such as Huq et al. (2005) that found an increase of 5 °C in water temperature resulted in a 3.31-fold increase in the risk of cholera. Collectively, the lack of reporting on the environmental conditions further limits the ability to determine variables that influence the occurrence and spread of V. cholerae in the environment. Understanding environmental conditions becomes more crucial, especially within the context of how climate change can affect the spread of cholera and other water-related diseases.

Details on sample preparation were also poorly reported. For example, while most studies used filtration, 45% of the studies did not report on their sample preparation method prior to analysis. A large proportion (79%) of the studies detected V. cholerae subgroup O1 or O139 in the relevant samples. There have been seven cholera pandemics in the past two centuries that were associated with V. cholerae subgroup O1 (biotypes ‘classic’ and ‘El Tor’) and O139 (Mutreja et al. 2011). V. cholerae O139 emerged in late 1992 as the predominant strain, displacing V. cholerae O1 El Tor (Ramamurthy et al. 2022). It was first identified in Bangladesh and India, spreading to neighbouring countries. Cholera outbreaks are reported to be only associated with V. cholerae O1 and O139 (Nasreen et al. 2022; CDC 2024).

Culture-based methods (n = 95) were the most widely used lab detection method for V. cholerae, followed by PCR (n = 68), immunological, and antibody detection (n = 57). Culture-based and/or PCR methods are regarded as the gold standards for clinical testing. However, for water and wastewater, the WHO recommends the use of multiple genes and sequences to confirm the presence of toxigenic V. cholerae and cholera epidemic concern (WHO 2024). This also eliminates the issue of viable but non-culturable V. cholerae in water and wastewater samples.

Cholera is characterized by the presence of virulent genes, such as those encoding cholera toxin and toxin-coregulated pilus, which play a crucial role in the pathogenesis of the disease. Numerous virulent strains were described in the studies, namely ctxA/B, tcpA, toxR, and rfb genes (Table 1). More than half of the studies (54%) determined the presence of the ctxA/B virulence gene, which causes severe watery diarrhoea which is a common symptom amongst those diagnosed with cholera (Takemura et al. 2017). A quarter of studies (25%), described the toxR gene which encodes a transmembrane protein that controls the transcription of toxin genes, pathogenesis, and virulence (Kopprio et al. 2017). There were other various virulence genes reported from 41% of studies, with these including rtxA, rtxC, OmpU, vpi, hylA, zot, and ace genes (see the Supplementary Material 4). Considering that the spread of cholera may be via contaminated water, numerous studies have investigated the presence of virulence genes in environmental samples (Yilmaz & Goluch 2021). This gives us an indication of the type of strain that is circulating in an area and its epidemic potential (Faruque et al. 2004). Cholera toxin is a key biomarker for the identification of toxigenic V. cholerae and it is therefore important to monitor the sample isolates for their ability to produce cholera toxin.

The studies illustrate that WES has been used in identifying water bodies as reservoirs for V. cholerae and serves as a potential tool for larger-scale population screening, particularly in the absence of individual testing capacities. In terms of integrating clinical data with WES, 40% of studies presented clinical data, enhancing the contextual understanding of waterborne disease dynamics. However, it is important to note that in resource-constrained settings, clinical testing data may not be available. The integration of clinical data with WES data can provide opportunities to address a broader range of public health issues. For example, Shah et al. (2023) found similar patterns of antibiotic resistance in environmental and clinical strains, with environmental bacteria acting as a favourable reservoir for resistant pathogens, including V. cholerae.

Overall, 34% of the studies were conducted in response to a cholera outbreak, underscoring the role of WES in detecting V. cholerae in outbreak settings. For instance, Kahler et al. (2015) detected toxigenic V. cholerae in freshwater systems 2 years after the start of the cholera epidemic in Haiti, revealing the persistence of the virus in the environment. The surveillance of wastewater, drinking water, and water bodies that act as reservoirs are particularly useful for monitoring ongoing epidemics, especially in those areas where flooding has significantly threatened the spread of V. cholerae in the environment (Bhuyan et al. 2016). However, to date there is limited information and experience with integrating WES into existing surveillance systems, highlighting a key research gap, and supporting the need to explore improved data management to enable quicker access to WES data (WHO 2024).

This scoping review has a few limitations. First, we did not conduct a critical appraisal of the studies even though it is optional for scoping reviews. Secondly, the studies were gathered from two databases which might increase the chances of missing potentially relevant articles. We did not find reports or guidelines published by governments or public health organisations from our search that met the inclusion criteria. This study did not include reporting that was published on a public-facing dashboard that monitored V. cholerae in wastewater, wastewater-impacted environmental waters, or drinking water. Again, all articles obtained from the search published in a language other than English did not meet the eligibility criteria. This review also notes a geographical bias from the reported studies predominantly originating from India and South-East Asia. This geographical bias limits the ability to generalize evidence of WES of V. cholerae in regions and determine its applicability to support the management of outbreaks. Nonetheless, this review provides a comprehensive mapping of the evidence of WES for V. cholerae.

This scoping review underscores the role of WES in V. cholerae detection and surveillance, particularly in endemic regions such as India, Bangladesh, and South-East Asia. With the majority of studies originating from South-East Asia and India, there is limited insight into the effectiveness of WES for V. cholerae in other regions. Overall, improving the turnaround time from sample collection to the availability of data, enhancing the reporting of environmental factors conducive to V. cholerae, and better integration of clinical case data could strengthen the potential of WES as a tool for cholera outbreak management and early warning systems. Further investment is needed to establish WES in low-resource settings, particularly those where WASH conditions are inadequate. WES has not yet been demonstrated as an early warning system for a cholera outbreak. The public health value of WES is more evident for identifying environmental reservoirs of V. cholerae, and providing information on disease patterns, particularly when there are limited resources available for clinical surveillance. Overall, this review highlights the gaps in available evidence to better understand the potential role of WES in detecting and monitoring waterborne diseases such as cholera.

Research reported in this article was supported by the South African Medical Research Council [grant number: 40118] with funds received from the Department of Science and Innovation, the ELMA Relief Foundation [grant number: 23-R0008], and the National Research Foundation [grant number: 138011]. TK, NB, and MM are partly supported by the Research, Evidence and Development Initiative (READ-It) project. READ-It (project number 300342-104) is funded by UK aid from the UK government; however, the views expressed do not necessarily reflect the UK government's official policies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.