Environmental diagnosis of schistosomiasis, a tropical disease affecting more than 250 million people globally, is still challenging, which limits efficient eradication plans. There is a crucial need for resolutive and highly sensitive environmental tools to improve disease control. However, a major obstacle is the inability of current methods, including environmental DNA (eDNA), to distinguish infectious parasite life stages. Here, we develop and validate an environmental RNA (eRNA) approach that enables the detection and absolute quantification of Schistosoma mansoni miracidia and cercariae directly from water samples. After identifying life stage–specific transcripts, we design specific ddPCR assays which are strongly specific to S. mansoni and to each life stage. Comparing with eDNA assays, laboratory experiments demonstrate that stage–specific eRNA assays accurately reflect the relative abundance of each life stage, detect nucleic acids released from organisms, exhibit detection limits tenfold lower than eDNA assays. Also, under laboratory conditions, RNA persists in water longer than DNA. Field validation at six endemic sites in Brazil confirms that eRNA outperforms eDNA and conventional snail surveys in detecting active presence of S. mansoni cercariae, which indicates schistosomiasis transmission risk to humans locally. By enabling active–stage discrimination in environmental monitoring and improving sensitivity (compared to eDNA), our study advances both fundamental understanding and applied surveillance of schistosomiasis transmission, supporting elimination initiatives in affected regions. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, 22-PEXO-0011, 18-EUR-0019, 10-LABX-41

Objetivo: Utilizar a linguagem R para identificar o perfil epidemiológico e a distribuição espaço-temporal dos casos de esquistossomose, helmintos intestinais e caramujos Biomphalaria em Minas Gerais, no período entre 2010 a 2023. Métodos: Os procedimentos realizados no R e RStudio foram: obtenção dos dados do SISPCE, depuração, união das bases anuais, identificação do perfil, distribuição temporal e espacial dos casos. Resultados: Foram obtidos 72.408 casos de esquistossomose em 247 municípios, com positividade média de 3,06%. Foram coletados um total de 213 B. glabrata, 66 B. straminea e 71 B. tenagophila. Em relação aos geo-helmintos, foram identificadas 62.057 pessoas com ascaridíase, 45.610 com ancilostomídeos e 11.913 com tricuríase. Conclusão: A linguagem de programação facilitou rápido acesso, processamento e análise dos dados. O script criado poderá ser utilizado em qualquer estado do Brasil, além disso, contribuirá para obtenção de resultados e identificação de áreas prioritárias para o controle e vigilância da esquistossomose.

The persistence of schistosomiasis as a serious public health problem can be attributed, in part, to the lack of more accurate diagnostic techniques, particularly in areas with low prevalence and low parasitic burden. Thus, an effective diagnostic tool with broad applicability for detecting active infections in both high- and low-prevalence settings, as well as for accurately monitoring cure after therapeutic interventions, represent an instrument of utmost importance for controlling disease transmission. Therefore, we propose evaluating different commercial tests for schistosomiasis diagnosis identified through a review of all tests registered to be used in Brazil by the Brazilian regulatory agency (ANVISA) and those registered by other regulatory agencies, as well as prototypes of tests under development. In addition, we propose the development and evaluation of molecular diagnostic methodologies for schistosomiasis using different biological samples (stool, blood, and urine) from individuals living in an endemic area. The Kato-Katz technique, with 18 slides per stool sample, will be used as the reference test for definition of schistosomiasis cases. The performance of the evaluated techniques will be compared with respect to sensitivity, specificity, accuracy, positive and negative likelihood ratios, agreement with the reference test, cost, time, and ease of execution. The effectiveness and feasibility of these diagnostic tests will be assessed to recommend their incorporation into the schistosomiasis surveillance actions of the Ministry of Health within the Brazilian Unified Health System (SUS) in the short to medium term, considering the conditions faced by surveillance programs within Primary Health Care.

Most populations of Biomphalaria glabrata are highly compatible with Schistosoma mansoni strains, making it challenging to identify low-compatibility models to host-parasite interaction research. This study evaluated a B. glabrata population with low compatibility with the LE strain (SmLE) as a model. First, pigmented low susceptibility (LS) and albino susceptible (S) snails were paired to produce three progeny groups: Crossings (CR) between LS × S, LS (LS × LS), and S (S × S). F1 snails were individually challenged with SmLE, and compatibility and reproductive parameters were analyzed. Second, mass crossbreeding (150LS + 150S) produced F1 snails, later challenged with SmLE. Unchallenged F1 snails were then used for new mass crossbreeding, either without (CR1) or with (CR2) the introduction of LS snails (1:1 ratio). The F2 snails were challenged, and compatibility was assessed. In the pairings, the CR snails were predominantly pigmented (CRp). At 8 miracidia/snail challenge, F1 snails compatibility followed the pattern S > CRp > LS, consistent in both pairings and mass crossbreeding. In F2, CR1 resembled S, while CR2 showed intermediate compatibility (S=CR1 > CR2 > LS). Although crossings led to a reduction in the compatibility of F1 snails compared to the S group, the results suggest that resistance transfer between LS and S B. glabrata populations is less effective than previously demonstrated for susceptible/compatible and resistant/incompatible populations of Biomphalaria tenagophila. Thus, only traditional snail control measures (e.g., environmental modifications) are advisable in transmission foci maintained by B. glabrata. This study demonstrates that the population tested is not suitable for snail control strategies, although it may serve as a valuable experimental model for investigating host-parasite interactions.

BACKGROUND:Schistosoma sp. transmission is linked to water bodies, poor sanitation, and the presence of intermediate hosts. Nevertheless, parasite detection in snails is hampered by challenges in snail sampling and low infection rates, mainly in moderate and low-endemic areas, as well as requiring specialized personnel and being time-consuming. Thus, there is a need to improve tools to assist schistosomiasis surveillance and an environmental DNA (eDNA) approach may help to overcome these limitations. Here, we standardized and used an eDNA-based approach to monitor Schistosoma mansoni occurrence in two schistosomiasis endemic areas from Minas Gerais, Brazil. METHODS:The eDNA approach was standardized for local conditions by evaluating the specificity of the qPCR assay in detecting the parasite DNA. Water from snail breeding tanks containing Biomphalaria glabrata, either infected or not with S. mansoni, was used to standardize the eDNA filtration and extraction protocols. Three molecular techniques- Low-Stringency PCR (LS-PCR), Loop-mediated isothermal amplification (LAMP), and quantitative PCR (qPCR)- were applied to investigate samples from snail tanks and two field surveys. Additionally, malacological surveys and measurements of water physicochemical and microbiological parameters were conducted at the same locations to know the species of mollusks present and the ideal environmental conditions to identify hotspots. RESULTS:The qPCR assay was specifically amplified Schistosoma sp. DNA without amplifying other trematodes presents in Brazil, ensuring accurate detection without cross-amplification. All three molecular assays efficiently detected S. mansoni DNA only from eDNA samples from tanks with infected snails. The eDNA approach, associated with LAMP and qPCR assays, successfully identified S. mansoni DNA at the same collection points where snails releasing cercariae were found and at one additional site, that was missed by traditional methods, underscoring its sensitivity. CONCLUSIONS:This study illustrates the potential of employing eDNA sampling combined with molecular techniques as an effective strategy for monitoring and identifying potential schistosomiasis transmission foci in endemic areas. This approach aligns with the WHO's roadmap for schistosomiasis elimination by 2030 and has implications for public health interventions and control measures.

Species distribution models (SDMs) are increasingly popular tools for profiling disease risk in ecology, particularly for infectious diseases of public health importance that include an obligate non-human host in their transmission cycle. SDMs can create high-resolution maps of host distribution across geographical scales, reflecting baseline risk of disease. However, as SDM computational methods have rapidly expanded, there are many outstanding methodological questions. Here we address key questions about SDM application, using schistosomiasis risk in Brazil as a case study. Schistosomiasis is transmitted to humans through contact with the free-living infectious stage of Schistosoma spp. parasites released from freshwater snails, the parasite’s obligate intermediate hosts. In this study, we compared snail SDM performance across machine learning (ML) approaches (MaxEnt, Random Forest, and Boosted Regression Trees), geographic extents (national, regional, and state), types of presence data (expert-collected and publicly-available), and snail species (Biomphalaria glabrata, B. straminea, and B. tenagophila). We used high-resolution (1km) climate, hydrology, land-use/land-cover (LULC), and soil property data to describe the snails’ ecological niche and evaluated models on multiple criteria. Although all ML approaches produced comparable spatially cross-validated performance metrics, their suitability maps showed major qualitative differences that required validation based on local expert knowledge. Additionally, our findings revealed varying importance of LULC and bioclimatic variables for different snail species at different spatial scales. Finally, we found that models using publicly-available data predicted snail distribution with comparable AUC values to models using expert-collected data. This work serves as an instructional guide to SDM methods that can be applied to a range of vector-borne and zoonotic diseases. In addition, it advances our understanding of the relevant environment and bioclimatic determinants of schistosomiasis risk in Brazil.

Improvements in diagnostics for schistosomiasis in both humans and snail hosts are priorities to be able to reach the World Health Organization (WHO) goal of eliminating the disease as a public health problem by 2030. In this context, molecular isothermal amplification tests, such as Recombinase Polymerase Amplification (RPA), are promising for use in endemic areas at the point-of-need for their accuracy, robustness, simplicity, and time-effectiveness. The developed recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) was used to detect S. mansoni DNA from both laboratory and field Biomphalaria snails. Laboratory snails were experimentally infected and used at one, seven, and 28 days post-exposure (dpe) to 10 S. mansoni miracidia to provide samples in the early pre-patent infection stage. Field samples of Biomphalaria spp. were collected from the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil, which are endemic for S. mansoni. The sensitivity and specificity of the SmMIT-RPA assay were analysed and compared with existing loop-mediated isothermal amplification (LAMP), PCR-based methods, parasitological examination of the snails, and nucleotide sequencing. The SmMIT-RPA assay was able to detect S. mansoni DNA in the experimentally infected Biomphalaria glabrata as early as one dpe to 10 miracidia. It also detected S. mansoni infections (55.5% prevalence) in the field samples with the highest accuracy (100% sensitivity and specificity) compared with the other molecular tests used as the reference. Results from this study indicate that the SmMIT-RPA assay is a good alternative test to be used for snail xenomonitoring of S. mansoni due to its high sensitivity, accuracy, and the possibility of detecting early pre-patent infection. Its simplicity and portability also make it a suitable methodology in low-resource settings.

The artificially created Lake Pampulha, within the metropolitan area of Belo Horizonte, Minas Gerais State, lacks detailed information about its freshwater mollusks, representing a challenge for the assessment and conservation of this ecosystem. In this study, conducted during June and August 2021, we collected specimens on four different occasions and at five sampling points around the lake, using different sampling equipment, such as a shovel and a scoop. During these collections, we identified, enumerated and measured 1538 species of mollusks and additionally examined the presence of trematode larvae. We identified twelve species of fresh water bivalves and gastropods: Biomphalaria straminea, Biomphalaria kuhniana, Biomphalaria occidentalis, Drepanotrema cimex, Pomacea maculata, Stenophysa marmorata, Physa acuta, Gundlachia ticaga, Melanoides tuberculata, Pseudosuccinea columella, Omalonyx matheroni and Corbicula largillierti. Echinostome and strigeocercaria types of larval trematodes were detected in B. straminea. Notably, some species of mollusks have not previously been recorded at Lake Pampulha. The analyses revealed differences in the composition and abundance of species, highlighting the higher number of mollusk species in areas more impacted by human actions. This study expands our understanding of mollusk diversity at Lake Pampulha, and provides valuable data for longitudinal comparisons of water quality and considerations of the conservation of native species. Furthermore, it highlights the importance of choosing appropriate sampling equipment, depending on the research objectives. The presence of invasive species of medical and veterinary relevance as intermediate hosts of parasites reinforces the need for efficient environmental protection strategies to preserve this artificial, aquatic environment widely used by the local population and by tourists.

ABSTRACTSchistosomiasis is a neglected tropical disease caused bySchistosomaparasites.Schistosomaare obligate parasites of freshwaterBiomphalariasnails, so controlling snail populations is critical to reducing transmission risk. As snails are sensitive to environmental conditions, we expect their distribution is significantly impacted by global change. Here, we leveraged machine learning, remote sensing, and 30 years of snail occurrence records to map the historical and current distribution of competentBiomphalariathroughout Brazil. We identified key features influencing the distribution of suitable habitat and determined howBiomphalariahabitat has changed with climate and urbanization over the last three decades. Our models show that climate change has driven broad shifts in snail host range, whereas expansion of urban and peri-urban areas has driven localized increases in habitat suitability. Elucidating change inBiomphalariadistribution – while accounting for non-linearities that are difficult to detect from local case studies – can help inform schistosomiasis control strategies.