Environmental DNA and next-generation sequencing for post-elimination surveillance of neglected tropical diseases in the Philippines and the Western Pacific Region

Abstract

Post-elimination surveillance remains one of the most difficult challenges in neglected tropical disease (NTD) control. As countries in the Western Pacific Region approach elimination targets, conventional diagnostic tools such as microscopy and antigen rapid tests, optimised for moderate-to-high prevalence settings, are insufficient to detect the rare, focal, or reintroduced transmission that characterises near-elimination settings. The WHO Global Report on Neglected Tropical Diseases 2025 identifies weak surveillance systems and insufficient diagnostic innovation as primary threats to sustaining gains toward the 2030 road map, yet no operational framework exists for deploying environmental genomic surveillance in resource-limited post-elimination settings. The COVID-19 pandemic demonstrated the transformative potential of next-generation sequencing (NGS)-based wastewater surveillance for population-level pathogen detection, yet environmental NGS remains substantially underused for NTDs. In the Philippines, where lymphatic filariasis and Schistosoma japonicum approach elimination while soil-transmitted helminthiases, foodborne trematodes and rabies persist at low, focal prevalence, climate-driven rainfall and flooding concentrate pathogen burden within specific watersheds, drainage systems and habitats. Effective surveillance therefore requires frameworks that go beyond assay sensitivity and specificity to address how surveillance objectives, low-prevalence conditions, sampling strategy, and resource constraints jointly determine what can realistically be detected. This narrative review synthesises evidence on targeted and metagenomic NGS across the three locations in which an NTD signal can be recovered: environmental matrices, vectors and intermediate hosts, and animal reservoirs, using lymphatic filariasis, schistosomiasis, rabies, and related NTDs in the Philippines as a case example. We organise diseases by transmission route and the resulting signal location, which together determine the sampling matrix and the appropriate NGS approach (metabarcoding versus shotgun metagenomics). We argue that the principal remaining gap is not detection but integration: calibrating the environmental signal to infection prevalence and linking it with vector, host and clinical data to guide programmatic decisions, aligned with the 2030 NTD road map.

1 Background

Many tropical diseases are now approaching elimination as a public health problem in countries such as the Philippines (PH) and across the Western Pacific Region. The WHO’s 2024 NTD progress report documents a 32% reduction in the global population requiring NTD interventions since 2010, but flags weak surveillance and insufficient diagnostic innovation as the main threats to sustaining these gains toward the 2030 road map targets (1). The Philippines alone accounted for an estimated 49.5 million people requiring NTD interventions in 2023, with co-endemic lymphatic filariasis (LF), Schistosoma japonicum, soil-transmitted helminthiases, food-borne trematodiases, rabies, and leprosy, and formal elimination validation for LF and schistosomiasis still outstanding (1). As prevalence declines, however, transmission becomes increasingly focal, seasonal, and sensitive to population movement and climate-driven pulses (e.g., intense rainfall and flooding) (2–4). Surveillance must therefore shift from routine case reporting toward systems that can detect rare, local, or reintroduced transmission early enough to guide targeted responses. The 2023–2024 WHO Gap Assessment Tool (GAT) identified diagnostics and monitoring and evaluation as priority areas needing critical action across multiple NTDs (5). This underscores the operational difficulty of surveillance for these diseases. We argue that environmental eDNA surveillance can help address this diagnostic and monitoring gap. Specifically, it provides what the gap analysis identifies as missing: a catchment-level signal that screens several agents at once and detects rare, focal or reintroduced transmission below the reach of routine case finding. The approach has been adopted widely since the SARS-CoV-2 pandemic (6–9). Established eDNA and mNGS applications mostly target high-burden or endemic pathogens shed in abundance (6, 7, 10). Near-eliminated NTDs instead present a low-abundance, focal detection problem, which demands different sampling and analytical strategies.

This narrative review aims: i) to define the surveillance problems routinely faced in the Western Pacific Region under low prevalence, using the Philippines as a case study; ii) to categorise the relevant diseases by transmission pathway, since this determines where the pathogen signal resides and therefore which matrix to sample (Figure 1). Additionally, we draw on how eDNA surveillance has been applied elsewhere, and where direct evidence is lacking, we reason from adjacent disease types or sample matrices; iii) to outline sampling strategies for eDNA surveillance together with their principal challenges. Our argument centres on environmental eDNA surveillance, anchored to the WHO mandate and to the community-wide sampling logic; we therefore touch briefly on conventional patient- and population-based methods. Where disease- and country-specific evidence was limited, we drew on the broader metagenomics and environmental-surveillance literature, since many methods discussed were demonstrated only as research tools and not originally developed for NTDs.

Figure 1

As a narrative review, literature was selected thematically rather than systematically, drawing on peer-reviewed work from 2015 to 2025 across five domains: NTD epidemiology and programmatic status in the Philippines and Western Pacific Region, conventional and molecular diagnostics; eDNA/RNA and NGS methods for wastewater, vectors and environmental matrices; sampling and statistical design for low-prevalence surveillance, and bioinformatics and implementation frameworks. Seminal pre-2015 methods papers and WHO technical reports, including the Global Report on Neglected Tropical Diseases 2025, were included where relevant.

2 Progress, residual transmission and surveillance challenges for neglected tropical diseases in the Philippines

In the Philippines, despite substantial control progress, several of these NTDs remain locally transmitted or persistently endemic, with a burden pattern dominated by vector-borne infections, including dengue (1). Ten NTDs were reported nationally in 2020-2023; six of these (lymphatic filariasis (LF), schistosomiasis, soil-transmitted helminthiases (STH), foodborne trematodiases, rabies and leprosy) were the main contributors to public-health burden (11, 12). Different NTDs are characterised by distinct transmission pathways (Figure 1). We group the NTDs by three transmission pathways: Environment-associated agents that shed into water or soil (e.g., schistosomiasis, STH, foodborne trematodiases); animal-reservoir agents that are maintained in non-human hosts (e.g., rabies); human-maintained agents that persist in human population and spread either through vectors such as mosquitoes (e.g., LF, dengue, and chikungunya) or directly from person-to-person without a vector (e.g. yaws, leprosy, trachoma, scabies). Leprosy and other contact-transmitted infections are spread by direct contact and leave no external signal to sample. They are therefore outside this scope.

3 Surveillance frameworks and diagnostic tools for NTDs

3.1 Two surveillance paradigms

In this review, we outline two main surveillance frameworks: The first, patient-based surveillance, relies on clinical specimens collected from both asymptomatic and symptomatic individuals, including those sampled through community surveys (13, 14). The second, environmental surveillance, draws on samples from water sources, vectors, intermediate-hosts and other reservoirs that aggregate signals from human-derived and animal samples (15, 16). For many NTDs, including LF, schistosomiasis and STH, WHO defines “elimination as a public health problem” as reducing incidence or heavy-intensity infection below agreed thresholds rather than completely interrupting transmission (1, 17). As mass drug administration (MDA) and complementary interventions reduce prevalence, infections become increasingly clustered in specific places and population groups, often accompanied by a substantial asymptomatic reservoir that routine case detection fails to capture (17). For these preventive-chemotherapy NTDs, the role of surveillance is distinct from supporting individual diagnosis and case management: it samples defined community populations rather than patients, as in WHO Transmission Assessment Survey (TAS) for LF, to guide decisions about when and where to scale up, sustain or withdraw interventions. Various statistical models have been developed to optimise the design of such community surveys, including sample size, the number of sites and acceptable error rates (18–20). Community DNA-surveillance using environmental samples applies the same community-level logic from the perspective of the sampled environment rather than the population (21). Both approaches target transmission at the population level rather than individual diagnosis, but by different routes: community surveys infer prevalence statistically from many individual tests, whereas environmental sampling recovers a signal already integrated within the matrix.

3.2 Conventional patient and population-based detection methods

Conventional patient-based surveillance remains central to clinical management and to the formal verification of elimination targets. Community-based surveillance instead samples defined populations irrespective of symptoms; for the preventive-chemotherapy NTDs (LF, schistosomiasis, STH), approaches such as the TAS guide stop-MDA and impact decisions. These approaches rely on clinical specimens and well-established diagnostic methods, including microscopy, Point-of-Care (POC) tests, and advanced tools such as antigen tests, serology and conventional PCR (14, 22, 23). WHO disease-specific guidelines for NTDs continue to prioritise microscopy, serological assays and targeted PCR or loop-mediated isothermal amplification (LAMP). Nucleic acid-based assays, including singleplex and multiplex qPCR or RT-qPCR, LAMP and transcription-mediated amplification (TMA), are now standard methods for many programme targets (6, 14, 15). Within this human-based framework, there is growing interest in more integrated platforms for the detection of multiple signals in a single panel. Examples include i) multiplex serological panels capable of measuring antibodies to multiple NTDs (24) and vaccine-preventable infections from a single dried blood spot (25), and ii) high-throughput or cartridge-based molecular systems that combine multiple pathogen targets in a single run to reduce per-test cost and laboratory workload (26). Recent work on integrated multiplex bead-based serology demonstrates that a single survey platform can measure antibodies to several NTDs and other infections simultaneously. When paired with geostatistical analysis, these data can reveal areas with a high probability of recent exposure, including transmission hotspots that were previously unknown to control programmes (27). Even patient-focused surveillance is consolidating multiple diseases onto single platforms, a trend that parallels the environmental NGS approaches below. Patient-based methods establish individual diagnosis and elimination validation; environmental NGS adds the catchment-level, multi-pathogen layer that low prevalence demands. This review focuses on the latter.

4 NGS applications in environmental surveillance of NTDs

4.1 Lessons from wastewater and environmental surveillance

Environmental surveillance complements patient-based systems by sampling reservoirs that integrate signals from many human and animal hosts, including those who never present to health facilities. This concept has been widely adopted for eDNA surveillance of human-settings, popularised during the SARS-CoV-2 pandemic (8, 28), and increasingly being used in environmental surveillance of aquatic pathogens (29), rare, endangered and invasive animal species in aquatic ecosystems (30), source-tracking of faecal contamination (31), and monitoring of biodiversity as ecosystem health indicator (32).

Even though in theory all approaches rely on the same principle in molecular genetics, transferring the approach to eDNA surveillance of NTDs, however, is not straightforward: SARS-CoV-2 offered a high-abundance, continuously shed signal from human populations connected to centralised sewers, detected against a single well-characterised genome (8, 28). This is in contrast to NTDs, which consists of a diverse set of pathogens typically low in abundance, with pathogen signals distributed across disease-specific matrices with very different properties (soil, rice field water, vectors, fish and snails; Figure 1, Table 1), rather than a single, often aquatic, waste stream (summarised in Table 2). Certain components of the toolkit under the WHO’s Wastewater and Environmental Surveillance (WES) guidelines (9) can be transferred to guide eDNA surveillance of NTDs: (i) internal/external standards, and (ii) controls to quantify detection limits. A third requirement, statistical treatment of non-detections (left-censored data and detection-probability models), derives from eDNA ecology and wastewater-epidemiology rather than the WES guidance (See Table 2). The principal non-transferable assumption is that of an abundant, continuously shed target confined to a single matrix.

4.2 Identifying disease transmission route and signal location

The first such adaptation is to locate the signal before sampling it. Identifying which NTDs in Philippines would most benefit from eDNA/NGS surveillance depends on both their epidemiological status and how their transmission is sustained, since the latter determines where the pathogen signal resides and whether it can be recovered. The location and recoverability of an environmental signal depends on the agent’s transmission biology and the ecology of its shed nucleic acids, including their production rate, physical state, transport pathway and environmental decay, which together determines which environmental matrix carries a detectable signal (15, 33). Drawing on national reports and the WHO’s Global Report on Neglected Tropical Diseases 2025 (1, 11, 34), the three routes introduced in Section 2 map to three signal locations (Figure 1). For eDNA surveillance, the transmission route must be paired with risk mapping, and the geographic distribution of infection, so that sampling is targeted to where the signal is both present and concentrated (37, 38). Together these factors define the sampling target: an environmental matrix (water, soil), a vector or intermediate host (mosquitoes, snails), or a reservoir host (animals). We propose this as the first-level organising principle for eDNA NGS surveillance, with transmission pathway determining the most appropriate location for signal recovery (Figure 1). Some agents intersect more than one category and several may share the same matrix and be screened together. For example, schistosomiasis is shed into water yet also involves a snail intermediate host, while a single reservoir host may carry several agents, as dogs can harbour multiple zoonotic parasites (36). These cases are captured in Table 1.

4.3 Prioritising NGS targets for neglected and nearly eliminated tropical diseases in the Philippines

Table 1 positions selected diseases within the framework based on the organising principles set out in Section 4.2, and summarises current eDNA applications, major gaps and opportunities for incorporation into surveillance in the Philippines. Where a use case is lacking (Table 1), we draw on adjacent work as inferential evidence.

From this mapping, eDNA surveillance can be applied in three ways, differing in what each setting most needs from it (the relative strengths of targeted assays and sequencing are discussed in Section 5.3). First, for infections at the verge of elimination (i.e., lymphatic filariasis (13)), the agent is near-singular (predominantly Wuchereria bancrofti, with a minor Brugia malayi focus, transmitted by mosquito vectors; Section 4.3.1). Here, eDNA NGS is best used sparingly on top of targeted PCR, qPCR and LAMP, mainly to refine residual foci and distinguish persistent local transmission from reintroduction by lineage (Table 1). Second, still widely-endemic soil-transmitted helminths (STHs), food-borne trematodiases, and S. japonicum (14) are ideal for NGS as they represent a diverse group of eukaryotic parasites that share conserved marker genes (e.g., cox1, ribosomal loci), are shed into the same environmental matrices (water, soil); and, for schistosomiasis and the foodborne trematodes, pass through overlapping intermediate hosts. A single multiplexed metabarcoding assay or mNGS can therefore recover many co-circulating agents at once and resolve them to species and lineage, with appropriate sampling strategy and choice of primers, mapping not just where contamination occurs but which lineages and which host-driven cycles sustain it. The feasibility of recovering many taxa from one conserved marker is shown by mitochondrial 16S eDNA, which resolved multiple vertebrate faecal sources in freshwater (31, 41). Third, for flood-linked zoonoses such as leptospirosis (42) and co-occurring AMR determinants (2, 43–46), the value is operational: because they can be added to the same environmental panels on top of NTD detection, allowing event-based, multi-disease surveillance around storms and seasonal peaks without separate sampling campaigns.

Together these define a tiered application logic. At near-elimination with a single dominant agent, sequencing is deployed sparingly atop targeted qPCR/LAMP, reserved for lineage-level discrimination of residual versus reintroduced transmission. Where multiple co-endemic eukaryotic parasites share environmental matrices and conserved marker loci, a single multiplexed metabarcoding panel (or mNGS) resolves them concurrently to species and lineage. Where the agent is host-maintained and not environmentally shed, surveillance remains on host-derived WGS rather than eDNA (Table 1).

4.3.1 Vector-borne: lymphatic filariasis

In the Philippines, LF has been eliminated as a public health problem in 44 of the 46 formerly endemic provinces, with ongoing transmission now largely localised within certain island regions (39, 47). The dominant agent, Wuchereria bancrofti, is mosquito-borne, human-maintained and has no significant animal reservoir (34, 39). Molecular xeno-monitoring by qPCR or LAMP on mosquito pools is WHO-endorsed as a post-validation surveillance strategy and serves as a proxy for community microfilaria prevalence (48–50). Few if any studies have applied metagenomic or metabarcoding approaches as xeno-monitoring tool for detecting W. bancrofti or Brugia spp., whether in the Philippines or globally. Mitochondrial and population genetic studies of W. bancrofti have resolved haplotype networks and infrapopulation structure, demonstrating that W. bancrofti diversity is low but connectivity between communities is high (52, 53). This is epidemiologically relevant since residual foci are then unlikely to be genetically isolated. Markers that resolve this variation could potentially distinguish local resurgence from a new introduction and trace transmission, but have rarely been applied. Conventional cox1 screening of field-collected mosquitoes has recovered filarial signal, including Brugia-like and W. bancrofti sequences (51–53) but relied on single-marker amplicon Sanger sequencing and was not able to resolve mixtures or species due to sparse filarial reference databases (51). On the other hand, shotgun mNGS of single mosquitoes resolved the whole community, the mosquito, its blood meal and its microbial and eukaryotic load (54–57), but sensitivity for any low-abundance target fell in a complex community.

By contrast, long-read metabarcoding has demonstrated use for identifying filarial parasites in reservoir hosts. For example, using a long-read COI metabarcoding platform, Huggins et al. resolved co-occurring filarioids in canine blood to genotype level, identifying Acanthocheilonema reconditum, a Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. ‘hongkongensis’, and outperformed the Knott’s test and conventional PCR (36, 58). The same approach has since grouped zoonotic filarioids by haplotype across settings, from Cambodian and Bhutanese dogs to the Sri Lankan reservoir, where the canine Brugia genotype is related to the one infecting people (36, 60–62). These studies show that long-read metabarcoding of conserved genes can recover lineage information from a mixed sample: separating co-occurring species, assigning genotype, and linking haplotype groups to infer transmission. The capability is therefore demonstrated; what remains is its transfer to the mosquito, where xeno-monitoring could move beyond detecting W. bancrofti to resolving lineage and origin. This lineage resolution is precisely the discrimination identified above as the principal value of sequencing at near-elimination: separating residual transmission from reintroduction.

4.3.2 Environmentally shed signal: schistosomiasis, STH, foodborne trematodes

Schistosomiasis, soil-transmitted helminths (STHs) and foodborne trematodiases are among the most widely endemic NTDs in the Philippines (Figure 1; Table 1) (11, 12, 37, 40, 64, 65). These diseases share a common transmission route: their transmissible stages are shed into water or soil and sustained by faecal/urinary contamination of a shared environmental compartment (i.e., water for schistosomiasis and foodborne trematodes, soil for STHs) (66–68). Transmission to humans occurs either directly through that compartment (skin penetration or ingestion of the infective stage) or indirectly through one or more aquatic intermediate hosts (e.g., fish/snails; Figure 1), with animal definitive hosts (e.g., water buffalo (69, 70)) acting in parallel to human as reservoirs that replenish the same environment; which can be further complicated by the lack of WASH under disaster-prone settings (71, 72). Conventional environmental surveillance of these agents therefore relies on a single principle: the matrix dictates which life stage can be recovered and identified by morphology (Table 1).

Schistosoma japonicum: Schistosoma japonicum remains endemic but increasingly focal, mainly in parts of Eastern Visayas and Mindanao, and is targeted for elimination under the Philippine Multi-Disease Elimination Plan (MDEP) 2024–2030 (40, 74). S. japonicum persists in irrigated rice fields, lakeshore and floodplain systems through its amphibious snail intermediate host Oncomelania hupensis quadrasi, and as a zoonosis is maintained in animal reservoirs such as water buffalos and cattle alongside human infection (65, 69, 75). Conventional surveillance of S. japonicum relies on morphological identification of a life stage, and the target differs by host, reviewed elsewhere (76). Molecular methods increasingly used in eDNA surveillance show improvement in sensitivity and species specificity across three distinct targets, each interrogating a different point in the cycle: the snail host, the animal reservoir, and the water body. PCR and LAMP detected pre-patent S. japonicum infection in crushed Oncomelania, before cercarial shedding (76, 77). In water, cox1 qPCR detected S. japonicum and O. hupensis quadrasi eDNAs shed into freshwater, first quantified down to half a cercaria-equivalent in spiked samples (78) and applied at Philippine field sites, where it matched snail-crushing and detected the snail where malacological survey found none (75); a companion study extended this to soil for the dry sites the amphibious snail occupies (79, 80). Most target cox1, the 28S or 18S rRNA genes, or repetitive elements, and outperform microscopy at low intensity. Because these assays converge on the same conserved markers (cox1, 28S, 18S), those loci can potentially serve directly as metabarcoding targets, allowing S. japonicum, its snail host, and co-endemic agents to be captured together in one multiplexed panel.

Soil-transmitted helminths (STHs): STHs (Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma spp.) remain widespread, transmitted directly through soil contaminated with human faeces, without any intermediate host (37). The main burden falls on children and other vulnerable groups, and several species have zoonotic reservoirs, with pigs maintaining Ascaris suum and dogs and cats (81). Conventional methods have relied on recovering eggs and larvae from soil, wastewater and sludge by flotation or sedimentation, followed by identification using microscopy (73, 82). Molecular techniques such as PCR followed by conventional sequencing, multiplex qPCR and digital droplet PCR (ddPCR) and LAMP have been increasingly demonstrated as alternatives to microscopy for detecting STHs in soil, wastewater, reclaimed water, drainage-ditch and stool (67, 71, 83–86). These molecular assays target one or more agents and generally give results comparable to microscopy (67). High-throughput NGS is far less established for STHs. Metabarcoding has been demonstrated for gastrointestinal nematodes from rodents targeting the 18S rRNA gene (87) and for nematodes in soil, although sparse reference databases (notably for cox1) limit species recovery (88, 89). Untargeted mNGS are also few, and those that exist were largely run on cultured eggs or stool rather than environmental matrices (90, 91). Metabarcoding and mNGS rest on the same principle as qPCR, that parasite DNA can be recovered from the source. However, the steep challenge is that parasite reads are heavily diluted by host and bacterial DNA in complex samples. Recent work has addressed this on two fronts. Analytically, a Strongyloides faecal-model validation found mapping to the high-copy parasite mitochondrial genome the most reliable strategy, with short-read outperforming long-read and bacterial contamination driving false positives (92). At sample preparation, hybridisation capture enriched Ascaris and Trichuris mitochondrial genomes from faecal DNA several thousand-fold over direct shotgun sequencing, recovering data from as few as tens of eggs per gram (93). As for S. japonicum and the foodborne trematodes, these shared markers (18S, cox1) and enrichment strategies position STHs for inclusion in the same multiplexed environmental panels once reference databases are sufficiently curated.

Foodborne trematodes: Food-borne trematodiases intestinal heterophyids (e.g., Haplorchis taichui, Stellantchasmus falcatus), echinostomes and Fasciola spp., acquired from raw/undercooked freshwater fish, crustaceans and snails in rice-fish, aquaculture and riverine systems, are widely endemic but under-reported in parts of the country (64, 94, 95); Opisthorchis viverrini has only recently been identified molecularly in Mindanao (94). Molecular methods have been applied across the same targets. In the Philippines, NGS and targeted PCR-sequencing have differentiated co-circulating Fasciola lineages in livestock (64, 70) and characterised heterophyid infections such as Haplorchis taichui in human stool (95), with fish-borne heterophyids recently identified molecularly in PH communities (94). Elsewhere, amplicon sequencing and mNGS have profiled trematode metacercariae in fish and snails and detected trematode eDNA in water (16, 96–98), and combined eDNA, molecular cercariometry and snail surveys to characterise trematode diversity (99). As with S. japonicum, these assays target conserved markers shared across the trematodes, so the same loci can anchor fish, snail and water eDNA metabarcoding panels, adapting regional pipelines to map foodborne-trematode hotspots in rice-fish and riverine systems and to evaluate aquaculture and food-safety interventions (16, 64, 96, 97). For the foodborne trematodes, two hosts are examined: snails for cercariae, and the second host (fish or crustacean) for metacercariae encysted in tissue, recovered by digestion and microscopy. Each of these methods is a morphological identification of a specific life stage, so each depends on intact organisms and trained microscopists and resolves poorly between closely related species (23).

4.3.3 Host-restricted signal: rabies and other animal-maintained zoonoses

Dog-mediated rabies is fully endemic, with human and canine cases reported across all regions and incidence highest in selected provinces despite expanding dog vaccination and post-exposure prophylaxis (PEP) coverage (101–103); domestic dogs are the principal reservoir of Lyssavirus rabies (100), and reservoirs for a range of other zoonoses, including zoonotic STHs and filarioids (36, 81), with occasional spillover involving cats and wild canids (100). In the case of rabies, humans are infected mainly through bites and saliva exposure (104). Current surveillance relies on bite-case reporting, animal brain testing, and host-based PCR/antigen assays. In the Philippines, Sanger and NGS (including whole genomes) of dog and other animal brain tissue have been used to define Phillipines-specific lineages and island-structured transmission (102, 103, 105), while globally, host-derived WGS/NGS has been widely applied to phylogeography, outbreak reconstruction, and surveillance workflows (100, 101). Notably, dogs are increasingly sampled directly as sentinels and reservoirs for a range of NTD-related (e.g., lymphatic filariasis and other filarioids) and non-NTD zoonotic pathogens (105), where host-derived NGS and metabarcoding approaches have proven highly informative for species-level characterisation and epidemiological mapping (102, 103, 105). Drawing on SEA4 lineage work, routine WGS of representative dog (and human) rabies viruses from all regions should be embedded and linked to animal-movement data for risk mapping, detect reintroductions, and resolve island-to-island spread (102, 103, 105), maintaining focus on host tissues (brain, saliva/skin) and blood samples rather than environmental eDNA, which has no role in current rabies control and remains technically low-yield (102, 103, 105).

5 Post-elimination surveillance of neglected and nearly eliminated tropical diseases: design, technical and computational challenges

5.1 Surveillance objective and sampling design

As illustrated in Figure 2, a well-considered eDNA surveillance strategy begins by identifying the target NTDs and the surveillance question (17, 38): whether it is to (i) confirm that the prevalence of a given NTD is below a programme threshold (118), (ii) provide early warnings of re-introduction or resurgence (19), or (iii) tracking changes in risk during floods and seasonal peaks (3) (Table 2). The surveillance question determines how many false negatives and positives are tolerable and how much precision is needed, and these considerations in turn defines the sampling matrix, location, frequency and sample size (7, 17, 38, 119). Selection of target agent requires understanding of its transmission pathway under local conditions, aligned with public health priorities: whether the disease agent is human-maintained, environment-associated or animal-associated, and by what route it is transmitted (as established in Section 4.2; Figure 1) (9, 15, 36, 55). Addressing these questions at the outset informs the sampling design (for example, grab vs composite sampling; Table 2), and whether the chosen matrix, sample collection and choice of assays can meet surveillance objective and target detectability. Table 2 outlines considerations for sampling design of eDNA surveillance of NTDs.

Figure 2

Detecting a low-abundance signal depends on several interdependent factors, not one alone: infection prevalence, sampling design, environmental conditions and assay performance. It also varies with the transmission routes discussed earlier, since local environmental situation and matrix characteristics determine how a sample must be taken to yield a detectable signal (15, 33, 38, 119). For environmentally shed agents, shedding rate, eDNA decay and hydrological dilution dominate (33, 38, 120, 121). For vector-borne, human-maintained agents such as LF, the relevant determinant is the infection rate in the mosquito vector, used in molecular xeno-monitoring as a proxy for nearby human infection (109, 122), though the estimate depends strongly on vector species, trapping method and mosquito sample size, and holds at community rather than household scale (109, 123–126). For agents with an animal-associated component, such as zoonotic B. malayi, detectability additionally depends on infection in the animal reservoir (36, 59, 108, 127), and on vectors that bridge animal and human hosts, so surveillance that samples only the human-vector compartment may miss reservoir-maintained transmission (127). Because precision falls as prevalence declines, sampling design must also exploit spatial structure to remain efficient at low endemicity (118, 128, 129). Overall, an eDNA surveillance is iterative: results from each round of sampling should revise the design of the next (Figure 2). Familiarity with a given surveillance objective, target signals, and sample matrix often determines the simplest workable methods, down to the simple choice of DNA extraction protocol, and each refinement serves to reduce bias.

5.2 Sample preparation and processing in low-prevalence and disaster-prone settings

In post-elimination and near-elimination settings, eDNA surveillance is essentially a low-abundance signal detection problem in which upstream sample preparation becomes the first bottleneck, regardless of the downstream molecular method (15, 130). In the remote-island and archipelagic Western Pacific, this is compounded by geographic isolation and climate-driven extreme weather, such as high frequency and unpredictable tropical cyclones and flooding (3, 4), which can disrupt both surveillance and sample collection, storage and transportation, affecting sample integrity. For low-biomass samples, concentration steps such as filtration, flocculation and ultrafiltration are often needed to capture rare targets from large volumes of wastewater, surface water or vector pools (131), and these feed into downstream extraction, enrichment, library preparation and sequencing (131–136), each step carrying its own limitations that require process optimisation (137, 138). Non-aquatic matrices demand different front-end handling: soil and sediment require mechanical lysis, such as bead-beating, to release DNA from the robust eggs and cyst walls of STHs, alongside removal of humic acids and other PCR inhibitors these matrices carry (139, 140). Host-derived material is different again, since stool and blood from human or animal reservoirs, snail intermediate hosts and vector tissue are comparatively DNA-rich but dominated by host and microbiota background, so extraction, and where needed target enrichment, must recover the pathogen signal (139–142). Because every step can introduce loss or bias, sensitivity and specificity at very low biomass depend jointly on sample integrity, storage, concentration extraction efficiency, sequencing depth, and the rigorous use of internal/external standards and negative controls to manage contamination and quantify detection limits (15, 45, 143). Targeted enrichment techniques, including amplicon panels, hybridisation capture, probe-based tagging and culture-based enrichment, are increasingly being applied and refined to recover rare pathogen genomes from complex matrices, to enhance NTD signal detection during sample processing (130, 144–149).

5.3 eDNA NGS: opportunities and limitations

For eDNA surveillance, marker availability and matrix choice jointly determine assay feasibility. If a marker exists, the choice is targeted assays (qPCR, digital PCR, LAMP) versus metabarcoding, depending on robustness, specificity, sensitivity, and detection of divergent species. If suitable markers are unavailable, the fallback includes mNGS, conventional microscopy, or population-wide human screening (Table 1).

Among NGS approaches, metabarcoding amplifies a marker gene shared across a group of related-taxa (8). Metabarcoding success depends on both the marker and the reference database. Universal primers under-amplify taxa with mismatches in the primer binding region, amplification bias skews read proportions, and resolution varies enough between loci that multi-marker panels are usually needed (116–118), with short barcodes often resolving only to genus or family (119, 120). Assignment is then bounded by the database: incomplete references leave sequences unassigned or matched to the nearest relative (121), and the same reads yield different communities under different databases (117). Curated regional databases outperform global ones (122). Both problems are taxon-specific, so NTD work needs dedicated markers, validated primers and purpose-built reference databases. Most NTDs with a validated qPCR and PCR marker are in principle candidates for metabarcoding, since the locus is known and characterised (137, 150–152). But a primer built to detect a single target is not the same as one that resolves a clade, and the markers, panel design and reference coverage required are taxon-specific, as the filarioid platform shows (58); each NTD would therefore need its own marker, primer validation and database building (151). Without a clear objective, executable workflow and proper calibration, metabarcoding is hard to justify: LAMP and qPCR detect presence faster and more cheaply, and although metabarcoding resolves lineage, direct comparisons between qPCR and metabarcoding have yielded mixed results, with relative sensitivity depending on the target and on the conditions set out in each study (150, 153, 154). Metabarcoding of NTD agents remains limited, though feasibility has already been demonstrated across several groups: 18S rRNA for helminths and protozoa in wild animals (155), cox1 for filarial worms in dog populations (58, 60), multiplex panels for Leptospira (42), etc. MinION metabarcoding of bulk mosquito samples has likewise reproduced Illumina-grade species profiles for vector surveillance, with ~93% concordance, demonstrating decentralised vector screening directly relevant to molecular xeno-monitoring (156).

Because metabarcoding requires a marker for PCR amplification and is subject to primer biases, it can be less effective in recovering highly divergent members of a group (152, 157), which are better resolved by targeted assays or genome skimming (low-coverage shotgun sequencing that assembles high-copy targets like mitochondrial genomes and rDNA without prior PCR) (91). Shotgun mNGS, by contrast, sequences most of the nucleic acids in a sample regardless of the taxonomic groups (45). It therefore supports broad multi-pathogen surveillance and pathogen discovery, including unculturable and unknown taxa, but loses sensitivity when the target is rare against a high background (158, 159). Single-assay shotgun eDNA sequencing of river water illustrates this breadth, recovering pathogens spanning viruses, bacteria and eukaryotes while reducing the primer and PCR bias inherent to amplicon methods, at the cost of sensitivity for scarce targets (160). In situations where background such as host-DNA is abundant, metabarcoding may be a better choice for detecting a low signal (58). To date, the use of mNGS and metabarcoding for eDNA surveillance remains limited, and is largely conceptual outside defined sample types such as clinical specimens/research (161). Where it has been applied to complex matrices in the context of NTDs, reads are mapped to reference genomes to identify pathogens and characterise diversity, as in genome skimming of helminths obtained from human stools (91, 162).

We argue that no single method is universally superior as the choice of approach depends on surveillance objective, sampling design and an understanding of the constraints linked to the biology, sample matrix and transmission route of the disease agent, discussed in earlier section; so the question is less which method is best than which is fit for a given purpose. A key interpretive limitation of eDNA and mNGS-based surveillance is that nucleic acid detection does not confirm pathogen viability: DNA bound to sediments, biofilms or historically deposited material can persist and yield positive signals that overstate current transmission risk (15, 163–165), an ambiguity that matters most at low endemicity, where each detection carries disproportionate weight. This can be partially mitigated by viability-discriminating pre-treatments (e.g., propidium monoazide [PMA] or ethidium monoazide [EMA]), pairing DNA with RNA-based assays (RT-PCR or metatranscriptomics) or life-stage-specific transcripts, and by corroborating environmental signals with vector, intermediate-host and clinical data rather than relying on single detection (15, 111, 121, 165, 166); we note, however, that these differ in maturity, PMA/EMA being well established for prokaryotes but demonstrated for some protozoan and helminth targets but with variable efficiency and limited standardisation in NTD matrices (164, 167), RNA assays offering a more direct but largely untested proxy at low biomass (155, 156), and cross-referencing across data sources the most robust safeguard currently available. We therefore argue that viability inference should be built into surveillance design from the outset, alongside the calibration and baseline studies (discussed in Section 6; Priority 1), rather than applied as a post hoc correction.

5.4 Mobile and portable tools; and practical implementation

Nanopore platforms such as the MinION combine long reads, which improve taxonomic resolution, with real-time analysis, low cost and portability, advantages demonstrated for eDNA metabarcoding and shotgun NGS as well as clinical sequencing (61, 63, 160, 170–172). These capabilities have made it a widely proposed tool for pandemic response and disease surveillance, including field deployment at the point of sample collection (173–175). We therefore examine its suitability for chronic NTD eDNA surveillance in the fragmented, hazard-prone Philippines (3, 4). We argue that the value of these platforms for chronic NTD surveillance lies in smaller regional laboratories, not field deployment. The reasons are practical: low capital cost, modest infrastructure and power needs, simple training, and suitability for low, intermittent throughput (176, 177). These suit a network of regional labs better than one national facility or true point-of-collection use. Decentralisation only helps where central-laboratory access is the bottleneck and a single facility is hard to justify. The Philippines meets these conditions. As a fragmented archipelago of more than 7,000 islands, frequently hit by typhoons, flooding and seismic hazards (4) that sever transport and disrupt laboratories, it is ill-suited to routing every sample to one facility.

Regional assessments across South and Southeast Asia indicate pathogen-sequencing capacity within the region is thin, under-used and slow; with a median of 0.12 laboratories per million population, only about half of available monthly capacity used, and a median of 18 days from specimen collection to reporting (176). The same survey found these systems heavily dependent on external funding and constrained by fragile procurement and supply chains for sequencing reagents and consumables (178). A scoping review of nanopore use across Africa reports the same pattern, under half of studies sequenced in-country, per-sample costs of US$10 – 71, heavy donor dependence and almost no routine diagnostic uptake, indicating that the bottleneck is access and sustainability rather than the technology itself (179). A parallel synthesis of eDNA practitioners reaches the same conclusion for environmental applications in Southeast Asia, specifically citing shortages of local bioinformatics expertise, few dedicated eDNA facilities, and the absence of curated regional reference databases and adaptable, locally-validated standards (151). Yet in-country sequencing can be made to work in such settings and to feed directly into disease control: in Cambodia, locally-executed metagenomic sequencing of febrile patients identified vector-borne and zoonotic pathogens and prompted changes to national surveillance (180).

Acute disease is different. For Ebola or Lassa fever, portable sequencing is justified by urgency and genuine field use during pandemic or emergency. A same-day result guides management and containment, and lineage data direct treatment and trace spread (173, 174). The NTDs here are chronic, so access to remote, low-prevalence settings, rather than urgency, is the main pressure toward the source. Because no same-day result is needed, that access is better served by decentralised regional capacity and sample transport than by field deployment (175, 181). Cost has shifted this calculus: environmental NGS, though variable with platform, provider, batching and depth, has fallen substantially, and while case detection stays cheaper per test (182, 183), sequencing is no longer prohibitive in the Western Pacific, especially with pooling or shared facilities. Environmental NGS does not replace case-based surveillance but adds a catchment-level layer screening several pathogens at once, conceptually similar to the WHO’s WES pilot (9), its value rising as incidence falls and individual testing yields less. Where even local capacity-building is impractical, regional referral networks and coordinated sample transport can pool low, intermittent demand onto shared sequencing and computing capacity, an approach proposed as the basis for sustainable mNGS programs in resource-scarce settings (180).

6 Research and implementation priorities

To date, eDNA surveillance for NTDs remains largely at a research and academic stage. It has not been demonstrated across the most important NTDs, nor consistently across settings, disease types and sample matrices. For neglected and nearly-eliminated tropical diseases, the question is therefore no longer whether detection is possible, but how to solve the upstream problems (Tables 1, 2) that determine whether a signal can be recovered at all, and then how to turn low-abundance, low-prevalence measurements into public health action. Here, we outline two research priorities for the adoption of NGS for eDNA surveillance of NTDs:

Priority 1: Calibration and baseline studies At present, calibration studies relating the environmental signal to infection prevalence remain lacking for most NTDs. The principal exception is qPCR-based molecular xeno-monitoring of mosquito pools, where estimated vector infection rates have been compared with human prevalence (109, 122). For sequencing-based metrics, no study to our knowledge has yet related read counts or normalised loads (from wastewater, open drainage, vector pools and eDNA/eRNA) to infection prevalence or transmission intensity in low endemic settings. eDNA NGS data are compositional (184): read counts give relative, not absolute abundance, so they confirm presence but not how much transmission is occurring, which is the information most monitoring programmes (e.g., Quantitative Microbial Risk Assessment; QMRA (185)) need to decide whether to intensify, sustain or withdraw effort. Calibration maps such a relative signal onto prevalence within a defined setting, letting a positive result be interpreted quantitatively and residual, low-level transmission be distinguished from a resurgent focus that warrants intervention (6, 10). qPCR can complement NGS by quantifying target abundance directly, as in the molecular xeno-monitoring of mosquito pools noted above; the complementary strength of NGS lies in discerning lineage information for targeted intervention. Calibration must be paired with baseline characterisation: establishing the background level of the signal in a given setting, together with its spatial and seasonal variation and the assay’s detection limit in that matrix, so that a later measurement can be read as elevated, declining or genuinely resurgent rather than as background noise (6, 33, 118). We therefore propose calibration and baseline studies as a funding priority, because without them programmes cannot act effectively on a positive eDNA result, especially in resource-stretched settings, where acting on an uncalibrated or relic signal diverts scarce resources from where transmission is genuinely occurring.

Priority 2: An integrated framework for low-biomass settings At low prevalence, clinical cases often go unreported or fail to present, especially in rural archipelagic settings like in the Philippines (118). The environmental signal then becomes, arguably, the primary evidence of ongoing transmission or persistence in hosts or the environment. Beyond the sampling considerations outlined in Figure 2 and Table 2, the research priority is to optimise every step of the workflow, since each step depends on the one before it having been tuned to retain the marker signal. This runs from establishing an appropriate surveillance objective to identifying the right matrix for the relevant signals, through the choice of DNA extraction protocol, to the development of suitable primers for metabarcoding (151, 186, 187). Integration then lies in linking signal data (lineage and co-occurring species from NGS; absolute quantification from qPCR) and environmental observations to clinical data, where available, to build a single picture of local transmission. This picture is then translated into simple operational guidance on how many samples, from which matrices, and over what time window are needed to trigger, pause or sustain intervention. Where clinical data exist, they help narrow the search, since prior knowledge of where human infection has occurred can focus environmental sampling on the most informative sites and improve the chance of recovering a signal at low-abundance (118, 128, 129). From an implementation perspective, priorities include embedding NTD-oriented environmental NGS within existing genomic surveillance platforms, harmonising sampling and metadata standards, and strengthening local bioinformatics and molecular epidemiology capacity, and securing the procurement and supply-chain systems on which sequencing depends, a recognised bottleneck across the region (151, 176, 178). That locally-run sequencing can translate into programmatic change has already been demonstrated in Southeast Asia (180). These steps are essential for countries such as the Philippines to move from small, externally-led pilots to sustainable, programmatically integrated use of NGS in post-elimination surveillance.

7 Conclusion

Post-elimination does not equate to pathogen extinction (1). The contribution of this review is to frame post-elimination NTD surveillance as a defined methodological sequence, from transmission pathway through signal location, matrix and method to calibrated interpretation, and to identify quantitative interpretation, rather than detection, as the current rate-limiting step. In the Philippines, LF and schistosomiasis and other neglected tropical diseases can persist or re-emerge through environmental, vector and animal pathways even when clinical case numbers are very low (12, 34, 34). Key priorities include refining detection limits and sampling designs for low-endemic settings, integrating multi-matrix NGS data with traditional indicators, and developing baseline and calibrated models that link environmental signals to human infection risk with explicit uncertainty. Experiences from the Philippines, with residual LF and schistosomiasis foci, and recurrent floods, provide a useful testbed for these approaches. As genomic surveillance capacity expands across the Western Pacific Region, environmental NGS should be embedded as a robust, interpretable and sustainable component of broader surveillance architectures and not an isolated research activity.

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