Laboratory and field validation of the recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) for snail xenomonitoring for schistosomiasis.

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.

Climate and urbanization drive changes in the habitat suitability of Schistosoma mansoni competent snails in Brazil.

Schistosomiasis is a neglected tropical disease caused by Schistosoma parasites. Schistosoma are obligate parasites of freshwater Biomphalaria snails, 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 competent Biomphalaria throughout Brazil. We identified key features influencing the distribution of suitable habitat and determined how Biomphalaria habitat 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 in Biomphalaria distribution - while accounting for non-linearities that are difficult to detect from local case studies - can help inform schistosomiasis control strategies.

FioSchisto's expert perspective on implementing WHO guidelines for schistosomiasis control and transmission elimination in Brazil.

The World Health Organization (WHO) recognizes schistosomiasis as one of the Neglected Tropical Diseases targeted for global elimination in the 2030 Agenda of the Sustainable Development Goals. In Brazil, schistosomiasis mansoni is considered a public health problem, particularly prevalent among vulnerable populations living in areas with poor environmental and sanitary conditions. In 2022, the WHO published a Guideline encompassing recommendations to assist national programs in endemic countries in achieving morbidity control, eliminating schistosomiasis as a public health problem, and advancing towards interrupting transmission. The perspectives presented here, collectively prepared by members of the Oswaldo Cruz Foundation's (Fiocruz) Schistosomiasis Translational Program (FioSchisto), along with invited experts, examine the feasibility of the WHO recommendations for the Brazilian settings, providing appropriate recommendations for public health policies applicable to the epidemiological reality of Brazil, and suggests future research to address relevant issues. In Brazil, the provision of safe water and sanitation should be the key action to achieve schistosomiasis elimination goals. The agencies involved in measures implementation should act together with the Primary Care teams for planning, executing, monitoring, and evaluating actions in priority municipalities based on their epidemiological indicators. Host snails control should prioritize judicious ecological interventions at breeding sites. The Information, Education, and Communication (IEC) strategy should be associated with water and sanitation and other control actions, actively involving school community. To identify infected carriers, FioSchisto recommends a two-stage approach of immunological and molecular tests to verify transmission interruption during the intervention and beyond. Praziquantel administration should be done under medical supervision at the Primary Care level. MDA should be considered in exceptional settings, as a measure of initial attack strategy in locations presenting high endemicity, always integrated with water and sanitation, IEC, and snail control. To assist decision-making, as well as the monitoring and evaluation of strategic actions, there is a need for an Information System. FioSchisto considers this systematization essential to make investments in strategic research to support the improvement of schistosomiasis control actions. Efforts toward schistosomiasis elimination in Brazil will succeed with a paradigm shift from the vertical prescriptive framework to a community-centered approach involving intersectoral and interdisciplinary collaboration.

Corrigendum: Assessment of the accuracy of 11 different diagnostic tests for the detection of Schistosomiasis mansoni in individuals from a Brazilian area of low endemicity using latent class analysis.

[This corrects the article DOI: 10.3389/fmicb.2022.1048457.].

Identification and characterization of microRNAs in Biomphalaria tenagophila and comparative analysis of their expression in Schistosoma mansoni-resistant and -susceptible snail populations.

BACKGROUND: Schistosomiasis is a neglected tropical disease caused by Schistosoma and affects over 240 million people worldwide. One of the most prominent causative agents is Schistosoma mansoni, which develops inside the intermediate host. Biomphalaria tenagophila is the second most important vector of schistosomiasis in Brazil and the Taim population is completely resistant to infection by S. mansoni. OBJECTIVE: This study aims to identify and characterize B. tenagophila microRNAs (miRNAs) and evaluate their differential expression in S. mansoni-susceptible and -resistant populations of B. tenagophila. METHODS: Two populations of B. tenagophila snails, susceptible and resistant to S. mansoni infection, were used to investigate the small RNA response of these snails after being infected with the parasite. Small RNA sequencing and quantitative real-time PCR were employed to identify and validate differentially expressed miRNAs. Bioinformatics analysis were performed to identify miRNA precursors and mature and evaluate their differential expression. FINDINGS: The study predicted 173 mature miRNAs and 123 precursors. Among them were six Lophotrochozoa-specific miRNAs, three mollusk-specific miRNAs, and six pre-miRNAs in a cluster. The small RNA sequencing and RT-PCR of B. tenagophila samples allowed assessing the expression patterns of miRNAs. MAIN CONCLUSIONS: The results obtained may support future studies in Biomphalaria spp., generating a global impact on disease control.

DNA barcoding as a valuable tool for delimiting mollusk species of the genus Biomphalaria Preston, 1910 (Gastropoda: Planorbidae).

Introduction: The genus Biomphalaria in Brazil includes 11 species and one subspecies, three of which are intermediate hosts of Schistosoma mansoni. Due to the recent evolution of this group, some species are difficult to identify based on morphological characters, making the use of genetic markers necessary for species identification. This study aimed to evaluate the use of partial sequences of the cytochrome c oxidase I (coi) gene for the identification of Biomphalaria species using phylogenetic reconstruction and species delimitation algorithms. The study tested the use of DNA barcoding technique for species delimitation within the genus. Methods: DNA barcoding was performed by sequencing a partial region of the coi gene from specimens, and the sequences were analyzed using phylogenetic reconstruction and algorithms to delimit Operational Taxonomic Units (OTUs). Results: The study found that the use of the coi gene in the reconstruction of the phylogeny of the genus might be an alternative for understanding the evolution and dispersion of species. However, this marker alone is not enough to solve complex taxonomic problems within the genus. A total of 223 sequences were analyzed, 102 of which could be separated using the barcode gap, enabling the correct identification of seven taxa. Discussion: The study demonstrated that accurate mollusk identification is necessary for effective schistosomiasis control. The DNA barcoding methodology was found to be promising for accurate mollusk identification, which is crucial for concentrating schistosomiasis control efforts in places where it is needed.

First report of Biomphalaria tenagophila (d'Orbigny, 1835) (Gastropoda/Planorbidae) in Pará State, Amazon region of Brazil.

INTRODUCTION: Mollusks belonging to Biomphalaria genus are intermediate hosts of Schistosoma mansoni. In the Pará State, Northern Region of Brazil, there are reports of B. glabrata, B. straminea, B. schrammi, B. occidentalis, and B. kuhniana occurrence. Here, we report for the first time the presence of B. tenagophila in Belém, capital of Pará state. METHODS: A total of 79 mollusks were collected and examined to search for possible S. mansoni infection. The specific identification was made by morphological and molecular assays. RESULTS: No specimens parasitized by trematode larvae were detected. For the first time the presence of B. tenagophila in Belém, capital of Pará state, was reported. CONCLUSION: The result increases the knowledge about Biomphalaria mollusks occurrence in the Amazon Region and specifically alerts on the possible role of B. tenagophila in schistosomiasis transmission in Belém.

Development of real-time and lateral flow recombinase polymerase amplification assays for rapid detection of Schistosoma mansoni.

Background: Accurate diagnosis followed by timely treatment is an effective strategy for the prevention of complications together with reducing schistosomiasis transmission. Recombinase Polymerase Amplification (RPA) is a simple, rapid, sensitive, and specific isothermal method with low resource needs. This research aimed at the development and optimisation of a real-time (RT) and a lateral flow (LF) RPA assay for the detection of Schistosoma mansoni. Methodology: Recombinase Polymerase Amplification reactions were performed at full- (as recommended) and half-volumes (to reduce costs), with RT or LF detection systems targeting the S. mansoni mitochondrial minisatellite region. The specificity was assessed using gDNA from other Schistosoma species, helminths co-endemic with S. mansoni, human stool, and urine, and Biomphalaria snail hosts. The analytical sensitivity was evaluated using serial dilutions of gDNA, synthetic copies of the target, and single eggs. The ability of both assays to detect the S. mansoni DNA in human urine and stool samples was also tested. The long-term stability of the RT-RPA reagents was evaluated by storing the reaction components in different temperature conditions for up to 3 weeks. Results: The RT- and the LF-RPA (SmMIT- and SmMIT-LF-RPA, respectively) presented similar results when used full- and half-volumes, thus the latter was followed in all experiments. The SmMIT-RPA was 100% specific to S. mansoni, able to detect a single egg, with a limit of detection (LOD) of down to 1 fg of gDNA and one synthetic copy of the target. The assay was able to detect S. mansoni DNA from stool containing 1 egg/g and in spiked urine at a concentration of 10 fg/µl. SmMIT-RPA reagents were stable for up to 3 weeks when kept at 19°C, and 2 weeks when stored at 27°C. The SmMIT-LF-RPA cross-reacted with Clinostomidae, presented the LOD of 10 fg and one synthetic copy of the target, being able to detect a single egg and 1 egg/g in a stool sample. The LOD in spiked urine samples was 10 pg/µl. Conclusion: The half-volume SmMIT-RPA is a promising method to be used in the field. It is specific, sensitive, robust, and tolerant to inhibitors, with a long-term stability of the reaction components and the real-time visualisation of results.

Assessment of the accuracy of 11 different diagnostic tests for the detection of Schistosomiasis mansoni in individuals from a Brazilian area of low endemicity using latent class analysis.

Background: Schistosomiasis is a parasitic disease associated with poverty. It is estimated that 7.1 million people are infected with Schistosoma mansoni in Latin America, with 95% of them living in Brazil. Accurate diagnosis and timely treatment are important measures to control and eliminate schistosomiasis, but diagnostic improvements are needed to detect infections, especially in areas of low endemicity. Methodology: This research aimed to evaluate the performance of 11 diagnostic tests using latent class analysis (LCA). A cross-sectional survey was undertaken in a low endemicity area of the municipality of Malacacheta, Minas Gerais, Brazil. Feces, urine, and blood samples were collected from 400 residents older than 6 years of age, who had not been treated with praziquantel in the 12 months previous to the collection of their samples. The collected samples were examined using parasitological (Helm Test® kit Kato-Katz), nucleic acid amplification tests -NAATs (PCR, qPCR and LAMP on urine; PCR-ELISA, qPCR and LAMP on stool), and immunological (POC-CCA, the commercial anti-Schistosoma mansoni IgG ELISA kit from Euroimmun, and two in-house ELISA assays using either the recombinant antigen PPE or the synthetic peptide Smp150390.1) tests. Results: The positivity rate of the 11 tests evaluated ranged from 5% (qPCR on urine) to 40.8% (commercial ELISA kit). The estimated prevalence of schistosomiasis was 12% (95% CI: 9-15%) according to the LCA. Among all tests assessed, the commercial ELISA kit had the highest estimated sensitivity (100%), while the Kato-Katz had the highest estimated specificity (99%). Based on the accuracy measures observed, we proposed three 2-step diagnostic approaches for the active search of infected people in endemic settings. The approaches proposed consist of combinations of commercial ELISA kit and NAATs tests performed on stool. All the approaches had higher sensitivity and specificity than the mean values observed for the 11 tests (70.4 and 89.5%, respectively). Conclusion: We showed that it is possible to achieve high specificity and sensitivity rates with lower costs by combining serological and NAATs tests, which would assist in the decision-making process for appropriate allocation of public funding aiming to achieve the WHO target of eliminating schistosomiasis as a public health problem by 2030.

A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil.

BACKGROUND: Schistosomiasis a neglected tropical disease  endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria. METHODS: Snails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples. RESULTS: Using the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88. CONCLUSIONS: Our findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor.

BACKGROUND: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. METHODS: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. RESULTS: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). CONCLUSIONS: The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

A multiplex PCR protocol for rapid differential identification of four families of trematodes with medical and veterinary importance transmitted by Biomphalaria Preston, 1910 snails.

Trematodes have complex life cycles with multiple hosts. Biomphalaria snails commonly act as the first intermediate hosts of several species that can affect human and animal health. The specific identification of larval trematodes found in snails is difficult and limited, since the taxonomy of these flukes is based on morphological traits of the adults found in vertebrates. Despite recent advances worldwide, studies aiming at the use of molecular tools for the identification of cercariae found in snails are scarce in the South America. In fact, most studies are focused on Schistosoma mansoni, with few efforts directed towards the identification of larvae of other parasites found in planorbids. When reported, these other parasites are identified as cercarial types, an artificial morphological system of classification. Therefore, alternative strategies for a correct, rapid and inexpensive identification of larval trematodes found in Biomphalaria are needed. This work aimed at developing a methodology capable of distinguishing four important families of trematodes (Clinostomidae, Echinostomatidae, Schistosomatidae and Strigeidae) commonly found infecting species of Biomphalaria. Using the rDNA sequences of 34 species as input for the online tool TipMT, we designed trematode family-specific primers targeting the ITS region optimized to be used in multiplex PCR. The panel of primers identified in this study was effective at the same PCR condition. The specificity of the primers was confirmed, and the PCR sensitivity ranged from 0.1 ng to 1 ag of the DNA of the parasite. This methodology was also effective for the detection of coinfection. Through a simple, fast, accurate, and inexpensive methodology, it is possible to properly identify the trematode families included in this study in a single PCR reaction. A family level identification provides important information about probable hosts, pattern of life cycle and possible impacts that the infection generates in a specific region, thus allowing the design of better control strategies, especially for those infections that have medical and veterinary importance.

Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata.

BACKGROUND: Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE: The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS: By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS: 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5' end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS: Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.

Schistosoma mansoni FES Tyrosine Kinase Involvement in the Mammalian Schistosomiasis Outcome and Miracidia Infection Capability in Biomphalaria glabrata.

Schistosomiasis is a neglected tropical disease (NTD) caused by helminthes from the Schistosoma genus. This NTD can cause systemic symptoms induced by the deposition of parasite eggs in the host liver, promoting severe complications. Functional studies to increase knowledge about parasite biology are required for the identification of new drug targets, because the treatment is solely based on praziquantel administration, a drug in which the mechanism of action is still unknown. Protein kinases are important for cellular adaptation and maintenance of many organisms homeostasis and, thus, are considered good drug targets for many pathologies. Accordingly, those proteins are also important for Schistosoma mansoni, as the parasite relies on specific environmental signals to develop into its different stages. However, the specific roles of protein kinases in S. mansoni biology are not well understood. This work aims at investigating the tyrosine-protein kinase FES (Feline Sarcoma) functions in the maintenance of S. mansoni life cycle, especially in the establishment of mammalian and invertebrate hosts' infection. In this regard, the verification of Smfes expression among S. mansoni stages showed that Smfes is more expressed in infective free-living stages: miracidia and cercariae. Schistosomula exposed to SmFES-dsRNA in vitro presented a reduction in movement and size and increased mortality. Mice infected with Smfes-knocked-down schistosomula exhibited a striking reduction in the area of liver granuloma and an increased rate of immature eggs in the intestine. Female adult worms recovered from mice presented a reduced size and changes in the ovary and vitellarium; and males exhibited damage in the gynecophoral canal. Subsequently, miracidia hatched from eggs exposed to SmFES-dsRNA presented changes in its capability to infect and to sense the snail mucus. In addition, the SmFES RNAi effect was stable from miracidia to cercariae. The establishment of infection with those cercariae reproduced the same alterations observed for the knocked-down schistosomula infection. Our findings show that SmFES tyrosine kinase (1) is important in schistosomula development and survival; (2) has a role in adult worms pairing and, consequently, female maturation; (3) might be essential for egg antigen expression, thus responsible for inducing granuloma formation and immunomodulation; and (4) is essential for miracidia infection capability. In addition, this is the first time that a gene is kept knocked down during three different S. mansoni life stages and that a tyrosine kinase is implicated in the parasite reproduction and infection establishment in the mammalian host. Accordingly, SmFES should be explored as an alternative to support schistosomiasis treatment and morbidity control.

Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata.

BACKGROUND: Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE: The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS: The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS: 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS: Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania amazonensis in skin samples.

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.

Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata

BACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.

Geographical distribution of intermediate hosts of Schistosoma mansoni in the states of Paraná, Minas Gerais, Bahia, Pernambuco and Rio Grande do Norte, Brazil, 2012-2014. / Distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni nos estados do Paraná, Minas Gerais, Bahia, Pernambuco e Rio Grande do Norte, 2012-2014.

OBJECTIVE: to describe the geographical distribution of intermediate hosts of Schistosoma mansoni in five Brazilian states. METHODS: this was a descriptive cross-sectional study; municipalities were selected in the states of Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) , and Rio Grande do Norte (98), for the period 2012 to 2014; these municipalities were chosen because they did not have current records of the presence of snails vectores de S. mansoni. The molluscs were captured and taxonomically identified and examined for S. mansoni cercariae. RESULTS: the work was carried out in 427 municipalities (99.5% of the 429 selected); the presence of mollusks was registered in 300 (70.2%) municipalities; Biomphalaria glabrata were found in 62 (21%) municipalities, B. straminea in 181 (60%), B. tenagophila in three (1%); B. glabrata/B. straminea association was found in 53 municipalities (18%) and B. glabrata/B. tenagophila association in one (0.3%) municipality. CONCLUSION: B. glabrata, B. straminea and B. tenagophila distribution records obtained in this study are consistent with previously known distribution.

Identification of Biomphalaria sp. and other freshwater snails in the large-scale water transposition project in the Northeast of Brazil.

The wide eco-bio-social intervention generated by the SaoFrancisco River Integration Project (PISF) may contribute to the dispersion or introduction of schistosomiasis intermediate hosts in areas without prior recording. The objective was to characterize the limnic malacofauna and its distribution along watersheds involved in the PISF. A cross-sectional study based on the collection of mollusks from 33 water bodies, from Aurora, Brejo Santo, Jaguaretama, Jaguaribara, Jati e Mauriti municipalities in the Ceara (CE) State was developed. The conchological characteristics were used to identify snails at the genus level. The snails of the genus Biomphalaria were analyzed for the presence of Schistosoma mansoni cercariae and the molecular identification (only mollusks from Brejo Santo-CE) for differentiation between species. The following species were found: Biomphalaria sp.; Drepanotrema sp.; Melanoides sp.; Physa sp.; and Pomacea sp. Pomacea sp. (75.8%) and Biomphalaria sp. (72.7%) were the most prevalent species. All municipalities showed Biomphalaria sp. Biomphalaria straminea (Porcos Stream) and Biomphalaria kuhniana was identified in the Boi 1 and Cipo reservoirs (Brejo Santo). The evaluated municipalities under the influence of the PISF present areas with potential for schistosomiasis transmission. It is necessary to intensify control actions and health surveillance in these areas.