Xenomonitoring surveillance of schistosome transmission: implications for schistosomiasis elimination and zoonotic transmission awareness

In Cameroon, despite numerous mass screening and treatment campaigns against schistosomiasis, reinfection persists, mainly due to continued contact with infested water bodies. Consequently, the disease remains a significant threat to population health and socioeconomic development. In this study, we assessed the environmental risk of schistosomiasis transmission by screening intermediate hosts. This complementary approach helps identify active transmission sites at human–water contact points and may help interrupt parasite transmission through targeted control interventions. We conducted a longitudinal study in Nkolbisson, a semi-rural area near Yaoundé, where mass drug administration campaigns have been implemented for more than a decade. Four human-water contact points were surveyed, and snails were collected and categorized by species. Infestation rates were determined using both parasitological techniques and molecular xenomonitoring with two primers. ETTS2/ETTS17 primers detected ribosomal DNA of Biomphalaria snails, Schistosoma species, and other trematodes, while SMF/R-F/SMF/R-R primers were specific to the Schistosoma mansoni 28S ribosomal region. Daily cercarial production was monitored in 34 infected Biomphalaria pfeifferi over eight days, and positive amplicons were sequenced and subjected to a nucleotide BLAST search for comparison with existing sequences. A total of 1,482 freshwater snails from four species were collected: Biomphalaria pfeifferi (361), Lymnaea natalensis (465), Physa acuta (348), and Melanoides tuberculata (134). Five morphologically distinct cercarial types were identified. Parasitological infestation rates were 19.59% for B. pfeifferi and 7.94% for L. natalensis. Molecular xenomonitoring revealed significantly higher infestation rates for B. pfeifferi: 45.14% with ETTS primers and 23.07% with S. mansoni -specific SMF primers. Infected snails produced a mean of 3151.25± 2165 cercariae per snail over eight days, with some individuals releasing over 2500 cercariae daily. Sequencing analysis confirmed the identity of S. mansoni, showing 100% sequence similarity with reference strains. The study identified active transmission foci where humans remain at high risk of S. mansoni infection and exposure to zoonotic trematodes despite years of mass chemotherapy. Molecular xenomonitoring proved to be more sensitive than traditional parasitological methods for detecting infected snails. It may consequently support targeted snail control strategies to complement mass drug administration for the sustainable elimination of schistosomiasis.

Human schistosomiasis results from infection with the larval forms of flat parasitic worms called schistosomes, which are released by freshwater snails serving as their intermediate hosts, and penetrate the human skin during contact with infested water (1). Elimination of schistosomiasis in sub-Saharan Africa remains a significant challenge. To date, no sub-Saharan African country has been validated by the World Health Organization (WHO) as having eliminated schistosomiasis as a public health problem (defined as <1% prevalence of heavy-intensity infections). Progress toward this elimination goal is impeded by incomplete mapping and impact assessments, ongoing transmission and persistent hot spots, difficulties in achieving adequate treatment coverage, and insufficient funding (2).

Where persistent transmission hotspots emerge during preventive chemotherapy campaigns, control of host snail populations can have a substantial impact, particularly when the program already achieves high treatment coverage levels. Co-implementation of snail control with mass treatment campaigns is expected to hasten the achievement of WHO goals for morbidity control and elimination as a public health problem. Snail control is thought to be essential to ultimately eliminate local transmission, in combination with Water, Sanitation, and Hygiene (WASH) interventions (3).

In Cameroon, despite numerous mass screening and treatment campaigns against schistosomiasis since 2012, the disease remains a significant threat to public health and socioeconomic development (4–9). Reinfection most likely occurs through contact with infested open water bodies, where infected snails release schistosome cercariae throughout the year (10, 11). The National Control Program has initiated, in some persistent hot-spot localities, chemical control of snail hosts in combination with improved WASH interventions. However, snail control activities remain limited and sporadic in several high-risk areas due to financial constraints. This situation, combined with population mobility, favors recurrent outbreaks and sustained transmission (4, 6, 7).

The 2021–2030 Road Map for Schistosomiasis and Soil-Transmitted Helminthiasis Elimination in Cameroon (12) calls for stronger alignment among stakeholders to prevent infections, emphasizing the need to establish routine monitoring systems and regularly update epidemiological databases to guide decisions on adapting or stopping Mass Drug Administration (MDA). By 2030, the roadmap aims to implement snail mapping and targeted snail control in selected endemic health districts, with active involvement of local communities. It also highlights that, as programs transition from morbidity control to elimination, tools must be adapted accordingly. Since interruption of transmission is the ultimate goal, particular attention should be given to detecting natural schistosome infections in snails and assessing the risk of human exposure to the cercariae.

Therefore, efforts are being made to improve the mapping of snail populations (13, 14) and to experimentally determine the appropriate lethal dose of Niclosamine for specific snail populations (15). Environmental surveillance through molecular xenomonitoring of intermediate snail hosts represents an innovative complementary strategy for identifying transmission hotspots and guiding targeted interventions (16). Traditional parasitological methods (cercarial shedding and snail squashing) used to detect trematode infections in snails, although useful, have limited sensitivity and may therefore underestimate the true risk of infection (14). Molecular xenomonitoring has shown considerable promise for detecting Schistosoma mansoni infections in Biomphalaria snails and can simultaneously identify other medically important trematodes (17, 18), thereby supporting the One Health approach, which integrates human, animal, and environmental health.

This study aimed to evaluate environmental transmission risks in a persistent focus near Yaoundé, Cameroon, where MDA has been implemented for over a decade. We employed both traditional parasitological methods and molecular xenomonitoring to: (1) identify and characterize human-water contact points harboring infected snail hosts; (2) compare the sensitivity of parasitological versus molecular techniques for detecting trematode infections; (3) quantify cercarial production rates to assess transmission intensity; and (4) characterize the diversity of trematode species present, including confirmation of human Schistosoma species through sequencing. The findings provide crucial insights for developing targeted snail control strategies and informing elimination efforts in similar endemic settings.

Study area

The study was conducted in Nkolbisson Health District, specifically in the Ekorezok Health Area, located approximately 7 km from Yaoundé, Cameroon (3°53′327″N, 11°27′412″E). This semi-rural area is characterized by an equatorial climate with four distinct seasons: two rainy seasons (from mid-March to June and from September to mid-November), with peak rainfall occurring in May and October, respectively, and two dry seasons (from July to August and from December to mid-March), with the driest period occurring in February. Annual temperature ranges from 17–30 °C with an average of 24 °C.

Ekorezok has been identified as a major transmission focus of intestinal schistosomiasis within the Nkolbisson Health District (19). The area is traversed by the Afeme River, which serves multiple community functions, including domestic activities, agriculture, and recreation. Despite access to improved water sources and sanitation facilities in most households, communities living near the river continue to use the river for various activities, maintaining exposure risk. The area has received regular MDA campaigns since 2012 as part of the national control program activities (Figure 1).

Study design and sampling sites

A longitudinal study was conducted over 3 months (February, March, and April 2024) with repeated malacological assessments. Traditional authorities were consulted to identify the specific human–water contact points regularly used by the local community. The rationale for the sampling period and sample size was based on the epidemiological objective of identifying active transmission sites within water bodies used by the population in the Ekorezok Health Area. Since schistosomiasis transmission depends on the presence of infected host snails, malacological surveys were strategically conducted during the dry season, a period previously reported in Cameroon and other endemic settings (6, 8, 20) to be associated with high snail abundance. February was specifically selected because it corresponds to the peak dry season in the district, when river water levels are at their lowest, leading to greater human-water contact and concentration of snail populations in limited aquatic habitats. Surveying during this period, therefore, increased the likelihood of detecting infected snails and accurately identifying potential transmission hotspots. Four contact points (designated A, B, C, and D) were selected based on the reported frequency of community use. Following a cross-sectional household-based survey conducted in the Nkolbisson Health District in 2018, the prevalence of schistosomiasis was estimated at 7.8% (Fossuo et al., unpublished data). In 2025, the WHO-ESPEN schistosomiasis endemicity report classified the district as having a moderate prevalence level (10–49%) (21).

Snail collection and identification

Snail sampling was conducted using standardized methods described by Madsen et al. (22). Two trained field collectors used a standard scoop net (0.2 mm mesh size) attached to a 2-meter metallic handle to systematically scrape aquatic and shoreline vegetation. Additional collection was performed using gloved hands in deeper areas, and tweezers for specimens on stones or plant supports (11). Sampling was conducted between 10:00 AM and 2:00 PM, with 15 minutes allocated per location per collector. Sampling areas extended 15–20 meters along the water body perimeters.

Collected snails were preserved in labeled 250-mL jars with site water and transported in coolers to the Laboratory of Parasitology and Ecology, University of Yaoundé I. Species identification was performed using conchological criteria (23).

Parasitological assessment

After two days of laboratory acclimation, intermediate host species (B. pfeifferi and Lymnaea natalensis) were subjected to cercarial shedding assays. Individual snails were rinsed and placed in well culture plates containing 1 mL filtered source water. At 10:00 AM, snails were exposed to a 100W electric bulb light for 3 hours to induce cercarial shedding, timed to coincide with peak shedding periods (24). Wells were examined under a dissecting microscope at 4× magnification for the observation and enumeration of cercariae. Non-shedding snails were re-exposed for up to 60 days.

Released cercariae were identified based on morphology, swimming behavior, and resting forms (25–28). Daily cercarial production was quantified in 34 randomly selected infected B. pfeifferi over eight consecutive days. Snails were maintained and fed fresh lettuce ad libitum. Following the observation period, snails were preserved in 70% ethanol for molecular analysis.

Molecular xenomonitoring

DNA extraction was performed on 208 B. pfeifferi specimens using the CTAB (cetyl trimethylammonium bromide) extraction protocol (14). Following DNA purification with 70% ethanol, DNA pellets were air-dried and resuspended in 50 μL distilled water, then stored at -20 °C until use.

PCR amplification employed two primer sets targeting conserved regions of ribosomal genes (29–31): (1) ETTS2/ETTS17 primers amplifying the highly polymorphic ITS1 region, enabling simultaneous detection of snail (600bp), schistosome (500bp), and other trematode DNA (1000bp); and (2) SMF/R-F/SMF/R-R primers specifically targeting the 28S ribosomal region of S. mansoni (350bp). Only specimens confirmed as B. pfeifferi by ETTS2/ETTS17 amplification, indicated by a 600 bp band, were included in the calculation of trematode infestation rates.

PCR reactions were performed in 20 μL total volumes containing: 15.35 μL distilled water, 1.25 μL 10× PCR buffer, 0.1 μL dNTPs mix (10mM), 0.25 μL each primer (ETTS2/ETTS17) or 0.25 μL each primer (SMF/R-F/SMF/R-R) (10 μM), 0.5 μL MgCl 2 (25mM), 0.05 μL Taq polymerase (5U/µL), and 2 μL template DNA from the extract. Thermal cycling conditions for ETTS2/ETTS17 were: initial denaturation at 95 °C for 45 s, followed by 34 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s, with final extension at 72 °C for 5 min. For SMF primers: initial denaturation at 94 °C for 20 s, followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at 65 °C for 20 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. PCR assays were validated using both positive and negative controls to ensure the reliability and specificity of the amplification results. Positive controls consisted of previously confirmed Schistosoma mansoni DNA samples, while negative controls contained PCR-grade water instead of template DNA to monitor potential contamination during amplification.

Expected amplicon sizes were: 600 bp for Biomphalaria spp., 500 bp for Schistosoma spp. and sister/hybrid species, 1000 bp for other trematodes, and 350 bp specifically for S. mansoni. Electrophoresis was performed on 2% agarose gels in 0.5× Tris-Borate-EDTA buffer with ethidium bromide staining. Electrophoresis was conducted at 110 V, 50 mA for 15 minutes, and gels were visualized under UV transillumination. Measures were implemented throughout the molecular procedures to minimize the risk of contamination during PCR analyses. DNA extraction, PCR preparation, and amplification were performed in separate working areas. Sterile filter tips and dedicated laboratory equipment were used, and reagents were handled carefully to avoid cross-contamination between samples. PCR assays producing weak, unclear, or ambiguous amplification profiles were repeated to confirm the reliability of the results.

DNA sequencing and analysis

Successful SMF amplicons were purified and quantified using a Qubit fluorometer with a 1× dsDNA working solution, yielding a final concentration of more than 4.0 ng/µL before sequencing. They were subsequently submitted for bidirectional sequencing using SMF/R-F (forward) and SMF/R-R (reverse) primers. Sequencing reactions were performed by the Centre for Research in Infectious Diseases (CRID) facility using an ABI 3500 XL sequencer. After trimming and sequence assembly, the obtained sequence lengths were 265 bp for the forward sequence, 251 bp for the reverse sequence, and 346 bp for the final contig. Raw chromatogram sequences were processed by the CRID service, which generated quality-checked AB1 and FASTA files.

Sequence analysis was performed using the Galaxy platform (https://usegalaxy.eu/). Reverse complement sequences were generated from the reverse reads using the RevSeq tool. These sequences were then aligned with the forward reads using the MAFFT tool to produce consensus sequences (32), which were subsequently compared with sequences available in the NCBI nucleotide database using the MEGABLAST algorithm (33).

Statistical analysis

Data were entered into Microsoft Excel 2007 and analyzed using SPSS version 20 (SPSS Inc., Chicago, IL, USA) and PAST statistical software (34) for the computation of descriptive statistics like snail abundances, infestation rates, and mean cercarial production. Chi-square tests were used to compare S. mansoni prevalences between detection methods, i.e., parasitological and molecular, and temporal variations. Statistical significance was set at α = 0.05 with 95% confidence.

Snail collection and species composition

A total of 1,482 freshwater snails representing four families, four genera, and four species were collected from the four human-water contact points over three months (Table 1). The species identified were: (i) Biomphalaria pfeifferi (Planorbidae, 361); (ii) Lymnaea natalensis (Lymnaeidae, 465); (iii) Physa acuta (Physidae, 348); and (iv) Melanoides tuberculata (Thiaridae, 134). The highest collection yield was recorded in April (n = 572; 38.6%, 95% CI: 36.1–41.1%), followed by March (n = 474; 32.0%, 95% CI: 29.6–34.4%) and February (n = 436; 29.4%, 95% CI: 27.1–31.7%). Total snail abundance did not differ significantly between February and March (χ² = 1.59, df = 1, p = 0.208). However, a significantly higher abundance was observed in April compared with both February (χ² = 18.35, df = 1, p < 0.0001) and March (χ² = 9.18, df = 1, p = 0.0024). Temporal analysis revealed distinct patterns in the different species. Biomphalaria pfeifferi (χ² = 51.38, df = 1, p < 0.0001) and M. tuberculata (χ² = 53.73, df = 1, p < 0.0001) populations showed progressively increasing abundance from February through April. In contrast, L. natalensis abundance significantly decreased (χ² = 149.92, df = 1, p < 0.0001) while Physa acuta remained stable (χ² = 0.02, df = 1, p = 0.8930) over the same period.

April recorded the highest B. pfeifferi abundance (n = 213; 37.2%, 95% CI: 33.3–41.2%) while L. natalensis was the predominant species in February (n = 225; 51.6%, 95% CI: 46.9–56.3%) and March (n = 189; 39.9%, 95% CI: 35.5–44.3%). Notably, M. tuberculata, a known competitor of Biomphalaria species, increased concurrently with B. pfeifferi abundance over the study period (Figure 2).

Characterization of human-water contact points

Spatially, the majority of snails were clustered at Point D, which harbored 1,308 specimens (88.3%, 95% CI: 86.7–89.9%), demonstrating an abundance significantly higher than all other contact points combined (χ² = 866.54, df = 1, p < 0.0001). This water contact site is used by local communities for multiple activities, including laundry, agriculture, washing, and swimming. It is characterized by slightly turbid water, a muddy substrate, and a stream-like water body, providing favorable conditions for intermediate host populations (Table 1). In contrast, Point C yielded 108 snails (7.3%, 95% CI: 6.0–8.6%), Point B yielded 42 snails (2.8%, 95% CI: 2.0–3.6%), and Point A yielded 24 snails (1.6%, 95% CI: 1.0–2.2%).

Parasitological assessment and cercarial diversity

Parasitological assessment conducted in B. pfeifferi and L. natalensis populations, selected for their capacity to host zoonotic trematodes, revealed natural infections only during March and April, whereas the infestation rate was null in February. For B. pfeifferi, the infection prevalence peaked at 29.0% in March (27/93; 95% CI: 20.7–39.1%) and remained relatively stable at 21.6% in April (46/213; 95% CI: 16.6–27.6%), suggesting specific seasonal transmission dynamics (Figure 3). Conversely, L. natalensis exhibited a low infection prevalence of 3.2% in March (6/189; 95% CI: 1.5–6.8%), which increased to 11.4% in April (10/88; 95% CI: 6.3–19.7%). The overall snail infestation rates were 19.59% (67/342) for B. pfeifferi and 7.94% (22/277) for L. natalensis. Infestation rates in B. pfeifferi varied significantly across collection periods (χ² = 16.33, df = 1, p < 0.0001), while L. natalensis also showed significant temporal variation between February and April (p < 0.0001).

Five morphologically distinct cercarial types were identified based on morphology and swimming behavior: echinostome cercariae, virgulate xiphidiocercariae, amphistome cercariae, S. mansoni cercariae type, and forked distoma cercariae. This last type of cercariae is morphologically recognized as avian schistosome cercariae, causative agents of cercarial dermatitis (“swimmer’s itch”) in humans. (Figure 3).

The dynamics of cercarial output were monitored over an 8-day experimental window using the infected B. pfeifferi cohort. The examined 34 infected B. pfeifferi shed a cumulative total of 89,626 cercariae, translating to a mean baseline output of 329.5 cercariae per snail per day. Surviving snails (n = 28 completing a full 8-day period) produced an average of 3,151.25 \pm 2,165 S. mansoni -type cercariae per individual, with total production values ranging from 132 to 10,804 cercariae per snail over the 8 days. Notably, highly productive individuals released over 2,500 cercariae within a single 24-hour cycle.

Molecular xenomonitoring of Schistosoma infections

The use of ETTS2/ETTS17 primers in 208 B. pfeifferi specimens highlighted distinct band sizes in agarose gels: a 600 bp band confirming B. pfeifferi species identity, a 500 bp band corresponding to schistosome DNA (DNA from different species of the genus Schistosoma), and 275 bp and 225 bp bands corresponding to non-schistosome trematode DNA. Meanwhile, the use of SMF/R-F/SMF/R-R primers yielded 350 bp bands corresponding specifically to S. mansoni DNA.

A total of 208 B. pfeifferi specimens, comprising 141 shedding-negative snails and 67 shedding-positive snails, were screened for snail species confirmation and characterized for schistosomes, other non-schistosome trematodes, and S. mansoni. Overall, 93.8% (195/208) of the screened snails were confirmed as B. pfeifferi by PCR. Of the 141 shedding-negative snails analyzed, 36.9% (52/141; 95% CI: 29.4–45.1%) were positive for schistosomes, 3.5% (5/141; 95% CI: 1.5–8.0%) for other non-schistosome trematodes (ETTS2/ETTS17 primers), and 15.6% (22/141; 95% CI: 10.5–22.5%) for S. mansoni (SMF/R-F/SMF/R-R primers).

Among the 67 shedding-positive snails examined, 41.8% (28/67; 95% CI: 30.8–53.7%) were positive for schistosomes, 4.5% (3/67; 95% CI: 1.5–12.3%) for other non-schistosome trematodes, and 34.3% (23/67; 95% CI: 24.1–46.3%) for S. mansoni. The infection prevalence in shedding-negative snails was statistically similar to that of shedding-positive snails for both schistosomes (χ²= 0.46, df = 1, p = 0.4976) and non-schistosome trematodes (p = 0.6983). However, the prevalence of S. mansoni infection was significantly higher in shedding-positive snails compared to shedding-negative snails (χ² = 9.53, df = 1, p = 0.0020).

This yielded an overall infestation rate of 45.13%, significantly higher than the parasitological rate of 19.59% (P< 0.001). Species-specific analysis using SMF/R-F/SMF/R-R primers detected 45 individuals with 350 bp bands corresponding to S. mansoni DNA, yielding a prevalence of 23.07%. This S. mansoni -specific rate remained significantly higher than parasitological detection (23.07% vs 19.59%, P< 0.05) but lower than the broader trematode detection achieved with ETTS primers (Table 2).

Molecular confirmation of species identity

Among the samples displaying the expected 350-bp PCR product, two amplicons were selected for sequencing. Both were successfully identified as S. mansoni. One sequence initially showed moderate-quality forward reads with overlapping peaks; however, repeat sequencing confirmed the species identification. The forward and reverse sequences were submitted to NCBI GenBank under accession numbers PZ235081 and PZ235082, respectively. The consensus sequence generated from both reads was subjected to BLAST analysis, which revealed 100% sequence identity with multiple S. mansoni reference sequences. The top matches included MK085971.1, AY157173.1, AY157256.1, MK085970.1, and OK310887.1. The alignment showed complete coverage across 346 nucleotides, with no mismatches or gaps (E-value = 6.43 × 10 -179; bit score = 640), thereby providing molecular confirmation of the presence of S. mansoni within the B. pfeifferi population. Additional high-scoring alignments (99.71% to 98.56% identity) with other African S. mansoni isolates further supported the species identification and demonstrated genetic consistency with regional parasite populations.

To the best of our knowledge, this is the first study to apply molecular xenomonitoring for schistosomiasis surveillance in Cameroon. Most schistosomiasis research conducted in Cameroon has primarily focused on determining the distribution and prevalence of infection within human populations (35, 36). While these human-based studies are essential for estimating disease burden, they do not directly capture transmission dynamics in the aquatic environment, limiting their ability to identify active transmission sites, estimate ongoing infection risk, and inform the most effective strategies for interrupting transmission.

The approach used in this study enables the identification of human–water contact sites associated with a high risk of infection within a persistent schistosomiasis focus. This strategy may facilitate, particularly in resource-limited settings, community awareness and engagement in vector control activities while allowing interventions to be more effectively targeted. This study demonstrates the persistence of active schistosomiasis transmission in a semi-rural area of Cameroon despite over a decade of mass drug administration. The identification of human-water contact points harboring infected intermediate hosts, coupled with quantitative assessment of transmission intensity through daily cercarial production, provides crucial insights for refining elimination strategies in endemic settings.

The observed dynamics of natural infestation between February and April indicate that snail infection likely occurred in February, as no active infections were detected in specimens collected during that month. Considering that the intramolluscan development of schistosomes typically requires approximately four weeks, depending on environmental conditions (37, 38), the infestation rates recorded in March and April likely reflect transmission events that occurred several weeks earlier. Given this result, we propose that the dry seasons, which are associated with increased human–water contact (36, 39), may represent a strategically important period for implementing MDA in this setting. Our interpretation is supported not only by the malacological data collected during the study period, but also by field observations of the local climatic and human behavioral patterns in the Ekorezok area. The study period corresponded to the driest months of the year in Ekorezok, during which human contact with open water sources markedly increases. During the rainy season, particularly in May, increased water levels, turbidity, and water instability reduce the use of these water bodies for bathing, laundry, recreational activities, and other domestic purposes. In contrast, these activities become much more frequent during the dry season, thereby increasing the risk of exposure to cercariae-infested water. Additionally, snail abundance was observed to be highest during the dry season, suggesting more favorable conditions for parasite transmission during this period. Our result, demonstrating the intensification of snail infestation in March and April, extends beyond this period as suggested by the high rate of infestation obtained with molecular markers and highlights the need to follow chemotherapy with a complementary targeted snail control intervention (37, 40).

The high infection rates observed in snails, compared with those reported in earlier studies (41), may reflect the current level of transmission in the human population, consistent with evidence showing a close relationship between human infection prevalence and snail infection rates (38, 40). Although concurrent human infection data were not collected in this study, the strongest evidence that schistosome infection rates in intermediate host snails are positively correlated with human prevalence comes from a recent systematic review and meta-analysis by Xin-Yao Wang and colleagues (40). They included 47 studies from 21 countries and found a statistically significant positive correlation between human schistosomiasis prevalence and infection rates in intermediate host snails (correlation coefficient r = 0.30, 95% CI: 0.01–0.50; P < 0.05). They concluded that snail infection data can serve as a useful indicator of transmission risk and should be integrated into surveillance and control programs.

The prevalence of schistosomiasis in Ekorezok may have increased in recent years due to the interruption of mass drug administration over two consecutive years, likely resulting from disruptions in the supply of Praziquantel previously supported by USAID. Our interpretation is based on longitudinal observations collected in the same study area (Ekorezok) over several years. During our previous study conducted in 2018 among 294 participants living in the Ekorezok health area, where praziquantel mass drug administration (MDA) campaigns were regularly implemented, the prevalence of human schistosomiasis was relatively low (7.8%) (Fossuo-Thotchum, pers. comm.). In contrast, in 2024, following the disruption of MDA activities, parasitological screening of Biomphalaria snails revealed an infection prevalence of 19%, suggesting intensified active transmission. Furthermore, our most recent snail screening conducted in March 2026, revealed a markedly higher Biomphalaria snail prevalence of 46% (A. Kengne Fokam, pers. comm.). Sustained preventive chemotherapy is recognized as essential for reducing transmission (37, 41).

While the infection rate of mollusks constitutes a key parameter in the epidemiological assessment of schistosomiasis, cercarial output per snail is a more direct indicator of transmission risk and/or potential disease spread, as it reflects the daily release of infective stages at human–water contact points (42). Accordingly, the high load of cercarial production observed in this study—higher than that reported in infected B. pfeifferi in previous studies (43)—may represent an important factor contributing to the persistence and amplification of transmission in this area. Moreover, the higher sensitivity of molecular xenomonitoring compared to traditional parasitological methods represents a key finding with significant implications for surveillance programs. The overall trematode prevalence detected using ETTS primers (45.14%) compared with that detected by cercarial shedding (19.59%) indicates that conventional parasitological methods substantially underestimate the true infection rates in snail populations. This enhanced sensitivity of molecular techniques has also been reported elsewhere (44) and likely reflects their ability to detect pre-patent infections, low-intensity infections, and infected snails that were not actively shedding cercariae at the time of examination.

The S. mansoni -specific prevalence of 23.07% detected using the SMF primers, although lower than that obtained with the broad-spectrum ETTS primers, still exceeded the prevalence estimated by parasitological methods. Sequencing of two representative PCR-positive amplicons confirmed their identity as S. mansoni, thereby validating the assay’s specificity. The obtained sequences showed 95.68% identity with multiple S. mansoni reference strains. Based on these results and the species-specific nature of the SMF primers, the remaining PCR-positive samples were assumed to correspond to S. mansoni.

Molecular xenomonitoring provides a highly sensitive approach for detecting schistosomiasis transmission by identifying parasite DNA in intermediate host snails, including pre-patent infections that cannot yet be detected through cercarial shedding. This improves early detection of transmission hotspots and provides a more comprehensive assessment of parasite circulation. However, PCR positivity does not necessarily indicate active transmission, as amplified DNA may originate from residual DNA in non-viable parasites, aborted infections. Although the use of species-specific primers, verification of expected amplicon sizes, and inclusion of appropriate controls minimize the risk of false-positive results, molecular findings should still be interpreted with caution. Therefore, PCR-based xenomonitoring is best viewed as a sensitive indicator of parasite exposure within snail populations, whereas cercarial shedding remains the reference method for confirming the presence of snails actively contributing to transmission.

A key novel aspect of our study is the detection of potentially zoonotic trematodes circulating in the same freshwater habitats as human schistosomes. The identification of five distinct cercarial types, including echinostome, amphistome, and distome cercariae, suggests complex transmission dynamics involving both human and animal parasites within shared aquatic ecosystems. Although investigations in humans and livestock were not conducted, these findings highlight the broader public health significance of these habitats and emphasize the importance of integrated One Health approaches combining environmental, human, and animal surveillance to better assess zoonotic transmission risks. Lymnaea natalensis, which showed 7.94% infestation rates, is known to transmit fasciolosis and other trematodes affecting both livestock and humans (45). The simultaneous presence of multiple trematode species in shared water bodies creates complex transmission dynamics requiring integrated control strategies.

From a programmatic perspective, these findings highlight several critical considerations for elimination efforts. First, the persistence of transmission despite years of MDA underscores the limitations of chemotherapy alone and supports WHO recommendations for complementary approaches such as targeted snail control (46). Second, the enhanced sensitivity of molecular detection suggests that conventional surveys may miss important transmission foci, leading to incomplete risk assessment.

The implications for elimination strategies are substantial. Environmental surveillance using molecular xenomonitoring could enable more precise identification of transmission hotspots, supporting targeted interventions such as focal snail control, environmental modification, or intensified chemotherapy. The One Health perspective highlighted by the identification of different cercarial types, including trematodes of potential veterinary and zoonotic importance, suggests opportunities for integrated approaches addressing both human schistosomiasis and zoonotic disease risks simultaneously.

The present study proposes an operational surveillance strategy to assess schistosome transmission risk in endemic settings using a combination of parasitological and molecular xenomonitoring. By detecting schistosome infections directly in field-collected snails, this approach moves beyond descriptive ecological studies and provides a practical framework for identifying active transmission sites and informing targeted control interventions. It’s successfully identified active transmission hotspots where communities remain at high risk of S. mansoni infection and zoonotic trematode exposure despite years of mass chemotherapy. Molecular xenomonitoring demonstrated superior sensitivity over traditional parasitological methods, with sequencing confirming species-specific detection of S. mansoni and multiple co-occurring trematode species. The high cercarial production load underscores substantial ongoing transmission potential, supporting the integration of environmental surveillance into national elimination programs for precise identification of transmission foci and targeted snail control strategies. Moving forward, molecular xenomonitoring should be considered a valuable complement to existing elimination strategies, particularly in MDA-insufficient areas, offering potential for more sustainable interventions that address environmental determinants of transmission through One Health approaches and community engagement in locally relevant control measures.

Limitations

This study has some limitations that warrant consideration. The three-month surveillance period (February-April 2024) may not capture complete seasonal transmission dynamics or represent annual patterns. Spatial sampling was restricted to four water contact points within a single health district, potentially limiting generalizability to other endemic settings. We acknowledge that environmental parameters can influence snail abundance, survival, and parasite transmission dynamics, and their inclusion would have provided additional ecological insight into habitat suitability and transmission patterns. We recommend that future studies incorporate detailed environmental and physicochemical measurements to characterize the ecological determinants of schistosomiasis transmission. Sequencing analysis was limited to selected positive samples rather than comprehensive genetic characterization of all detected parasites, and phylogenetic analysis was restricted to BLAST comparisons without detailed evolutionary or population genetic assessment.