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.

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.].

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.

Angiostrongylus cantonensis (Nematode: Metastrongyloidea) in molluscs from harbour areas in Brazil.

Angiostrongylus cantonensis is the most common aetiological agent of human eosinophilic meningoencephalitis. Following a report indicating the presence of this parasite in Brazil in 2007, the present study was undertaken to investigate the presence of A. cantonensis in the surrounding Brazilian port areas. In total, 30 ports were investigated and the following molluscs were identified: Achatina fulica, Belocaulus sp., Bradybaena similaris sp., Cyclodontina sp., Helix sp., Leptinaria sp., Melampus sp., Melanoides tuberculata, Phyllocaulis sp., Pomacea sp., Pseudoxychona sp., Rhinus sp., Sarasinula marginata, Streptaxis sp., Subulina octona, Succinea sp., Tomigerus sp., Wayampia sp. and specimens belonging to Limacidae and Orthalicinae. Digestion and sedimentation processes were performed and the sediments were examined. DNA was extracted from the obtained larvae and the internal transcribed spacer region 2 was analysed by polymerase chain reaction-restriction fragment length polymorphism after digestion with the endonuclease ClaI. Of the 30 ports investigated in this study, 11 contained molluscs infected with A. cantonensis larvae. The set of infected species consisted of S. octona, S. marginata, A. fulica and B. similaris. A total of 36.6% of the investigated ports were positive for A. cantonensis, indicating a wide distribution of this worm. It remains uncertain when and how A. cantonensis was introduced into South America.

Hybridism between Biomphalaria cousini and Biomphalaria amazonica and its susceptibility to Schistosoma mansoni.

Molecular techniques can aid in the classification of Biomphalaria species because morphological differentiation between these species is difficult. Previous studies using phylogeny, morphological and molecular taxonomy showed that some populations studied were Biomphalaria cousini instead of Biomphalaria amazonica. Three different molecular profiles were observed that enabled the separation of B. amazonica from B. cousini. The third profile showed an association between the two and suggested the possibility of hybrids between them. Therefore, the aim of this work was to investigate the hybridism between B. cousini and B. amazonica and to verify if the hybrids are susceptible to Schistosoma mansoni. Crosses using the albinism factor as a genetic marker were performed, with pigmented B. cousini and albino B. amazonica snails identified by polymerase chain reaction-restriction fragment length polymorphism. This procedure was conducted using B. cousini and B. amazonica of the type locality accordingly to Paraense, 1966. In addition, susceptibility studies were performed using snails obtained from the crosses (hybrids) and three S. mansoni strains (LE, SJ, AL). The crosses between B. amazonica and B. cousini confirmed the occurrence of hybrids. Moreover, hybrids can be considered potential hosts of S. mansoni because they are susceptible to LE, SJ and AL strains (4.4%, 5.6% and 2.2%, respectively). These results indicate that there is a risk of introducing schistosomiasis mansoni into new areas.

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.

Occurrence of Biomphalaria cousini (Mollusca: Gastropoda) in Brazil and its susceptibility to Schistosoma mansoni (Platyhelminths: Trematoda).

In Brazil, there are three intermediate snail vectors and two potential hosts of Schistosoma mansoni. Previous studies showed three variant molecular profiles to B. amazonica and evidenced intraspecific variations using sequence data. In this context, the aim of this study was to verify whether such differences would correspond to either B. amazonica or B. cousini. The snails were morphologically identified; PCR-RFLP and sequencing were carried out. Besides, B. cousini were submitted to susceptibility experiments to S. mansoni. Noteworthy, morphological data of Brazilian specimens predominantly showed the morphology described for B. amazonica. Nevertheless, PCR-RFLP results exhibited three variant molecular profiles for the specimens previously identified as B. amazonica and the phylogenetic analyses showed two groups one to B. amazonica and another to B. cousini. Furthermore, B. cousini showed to be susceptible to S. mansoni. These results confirm the occurrence of B. cousini in Brazil and points to the risk of introduction of schistosomiasis mansoni into new areas.

Preliminary studies investigating the occurrence of Biomphalaria cousini in Brazil.

Specific genetic profiles of Brazilian Biomphalaria species were previously standardized by molecular taxonomy through the analysis of restriction fragments, which were generated by digesting the internal transcribed spacer region of rDNA with the DdeI endonuclease. Biomphalaria amazonica displayed three distinct profiles. To investigate these distinct profiles, the same molecular technique, polymerase chain reaction and restriction fragment length polymorphism, was used with different endonucleases. In addition, morphological data were also used to compare B. amazonica specimens that were collected from Brazil, Colombia and Bolivia. The morphological characters of Bolivian molluscs were similar to B. amazonica, displayed a molecular profile of five restriction fragments and morphological data, whereas the Colombian mollusc population showed morphological characters similar to Biomphalaria cousini and a molecular profile of three restriction fragments, similar to B. cousini. The Brazilian specimens showed the B. amazonica and B. cousini molecular profiles as well as a third profile, which resembled a combination of the Colombian and Bolivian molecular profiles.

Epidemic of unilateral panuveitis in children from Brazilian Amazonia: clinical and etiological aspects in seven patients.

OBJECTIVES: To describe clinical presentation and results of diagnostic and therapeutic procedures in seven children from an epidemic of panuveitis in the Brazilian Amazonia, as well as environmental analysis and etiological aspects involved. METHODS: Patients underwent full pediatric and ophthalmic examinations, B-scan, ultrasound biomicroscopy, and serological tests. Ocular samples were thoroughly analyzed, including two enucleation specimens. Environmental investigation encompassed water, soil, and river fauna. RESULTS: All patients had bathed in the waters of a regional river, the Araguaia. Six of them presented with intermediate uveitis, with snowbanking. Five had cataract and four showed inferior endothelial opacity, with localized anterior synechiae. One showed total leukoma, with flat anterior chamber. Only two had active uveitis, one of them with anterior chamber nodule. Serology revealed high prevalence of anti-Toxocara canis immunoglobulin G (IgG) antibodies. In three cases, vitreous and lens samples disclosed spicules of freshwater sponges Drulia uruguayensis and D. ctenosclera, also detected in the waters of the river. CONCLUSION: Freshwater sponge spicules could be potential new etiological agents of ocular pathology, but further studies are needed, considering the heterogeneity of the ocular lesions and results of serological and environmental studies.

Biomphalaria molluscs (Gastropoda: Planorbidae) in Rio Grande do Sul, Brazil.

The present study was aimed at characterising Biomphalaria species using both morphological and molecular (PCR-RFLP) approaches. The specimens were collected in 15 localities in 12 municipalities of the southern region of the state of Rio Grande do Sul, Brazil. The following species were found and identified: Biomphalaria tenagophila guaibensis, Biomphalaria oligoza and Biomphalaria peregrina. Specimens of the latter species were experimentally challenged with the LE Schistosoma mansoni strain, which showed to be refractory to infection.

[Schistosomiasis in an ecotourism area in Minas Gerais State, Brazil]. / Esquistossomose em área de ecoturismo do Estado de Minas Gerais, Brasil.

This paper discusses schistosomiasis transmission in São José da Serra, a village with a population of 500 in the county of Jaboticatubas, Minas Gerais State, Brazil. The area receives thousands of visitors a year for ecotourism. The study was motivated by a case of acute schistosomiasis involving a couple that spent the 2007 Carnival (Mardi Gras) holiday in the area. Stool tests from 268 local residents (53.6% of the population) showed that 35 (13%) were positive for the infection. A comparison with a previous survey (2005) in the same location showed an increase in the schistosomiasis-positive rate from 9.6% to 12.5%, among the 56 individuals who participated in both surveys. A malacological survey of 65 Biomphalaria glabrata snails showed one specimen (1.5%) eliminating cercariae. In a similar survey in 2005, no positive snail specimens were found. The study indicates that active schistosomiasis transmission is occurring in the area, and that integrated educational programs are needed for both the local community and tourists.

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.

Prevalence and distribution of Angiostrongylus cantonensis (Nematoda, Angiostrongylidae) in Achatina fulica (Mollusca, Gastropoda) in Baixada Santista, São Paulo, Brazil.

INTRODUCTION: Angiostrongylus cantonensis is causes eosinophilic meningoencephalitis in humans. Worldwide expansion of this nematode is linked to the dispersion of their hosts. This study aimed to determine the prevalence of A. cantonensis infection in Achatina fulica in the nine municipalities that make up Baixada Santista, São Paulo, Brazil. METHODS: Angiostrongylus cantonensis larvae were analyzed using optical microscopy. We performed polymerase chain reaction and restriction fragment length polymorphism using restriction endonuclease ClaI, directed to the internal transcribed spacer region 2 of A. cantonensis larval DNA. RESULTS: Of the 540 snails analyzed, 117 (21.7%) were infected by A. cantonensis. For morphological and morphometric analyses, 60 larvae were used. Second-stage larvae were, on average, 358.2µm long and 26.4µm wide, while third-stage larvae were, on average, 450µm long and 21.12µm wide. The tails of the larvae ended in a fine tip. CONCLUSIONS: All municipalities comprising Baixada Santista had A. fulica that were naturally infected with A. cantonensis. All of the observed characteristics were typical of the species.

Genetic variability of Brazilian populations of Lymnaea columella (Gastropoda: Lymnaeidae), an intermediate host of Fasciola hepatica (Trematoda: Digenea).

In Brazil, Lymnaea columella is the most important intermediate host of Fasciola hepatica, the etiological agent of fasciolosis, which is a parasitic disease of veterinarian and human importance. Random amplified polymorphic DNA (RAPD) was used to investigate the genetic variability within and among nine Brazilian populations of L. columella comprising 205 individuals. A number of four primers were used for analysis of molecular variance (AMOVA). Out of 83 RAPD markers, 63 (76%) were polymorphic and revealed 119 unique RAPD profiles. The levels of genetic variability found in the populations were low and most of the genetic variation was interpopulational (81.6%) when compared to intrapopulational variability (18.4%). These results are in accordance with the dynamics and distribution of the populations analyzed.

Characterization of South American Snails of the Genus Biomphalaria (Basommatophora: Planorbidae) and Schistosoma mansoni (Platyhelminthes: Trematoda) in Molluscs by PCR-RFLP.

The identification of snails of the genus Biomphalaria can be done using morphological characteristics which depends on the size of the snails and skill and knowledge of researcher. These methods sometimes are not adequate for identification of species. The PCR-RFLP, using the ITS region of the rDNA, has been used to identify Brazilian species of the genus Biomphalaria. Nevertheless, there is a lack of information about snails from other Latin American countries. In addition, some snails may be infected by Schistosoma mansoni and when submitted to PCR-RFLP they show molecular profiles different from those previously standardized for the other mollusc species. In this work the molecular profiles of 15 species and the subspecies were established by PCR-RFLP of ITS-rDNA with the enzyme DdeI. Moreover, the molecular profiles of host species, B. glabrata, B. straminea, B. tenagophila, and B. prona, infected by S. mansoni were also established. The molluscs were dissected to permit morphological identification. These results contribute to a correct identification of snails of the genus Biomphalaria and detection of these snails infected by S. mansoni.

[Characterization of printed educational materials about schistosomiasis used in health education in endemic areas in Brazil]. / Caracterização de materiais educativos impressos sobre esquistossomose, utilizados para educação em saúde em áreas endêmicas no Brasil.

OBJECTIVE: to characterize printed educational materials about schistosomiasis produced at federal, state and municipal levels in Brazil. METHODS: the educational materials were characterized considering the following categories: 'format', 'parasite and intermediate host', 'definitive host (ill)' and 'disease'. RESULTS: 60 materials were assessed, three had no information about risk activities and 41 indicated more than one popular name for the disease, thus allowing greater reach among the target audience in diverse endemic areas; the biological cycle was missing or incorrect in 53 materials; the intermediate host (snail) was incorrectly illustrated, with use of stereotyped images in 39 and no image in one material; diagnosis was mentioned in 36 materials. CONCLUSION: the printed educational materials assessed had incorrect content which may compromise health education efforts; little attention was paid to schistosomiasis diagnosis.

Ontogeny and morphological variability of shell in populations of Leptinaria unilamellata (d'Orbigny, 1835) (Mollusca, Pulmonata, Subulinidae).

BACKGROUND: Recent studies concerning species of land snails have revealed that the shell morphometrics can provide evidence of the differentiation among populations. In many cases, the morphologic analysis combined with the investigation of molecular variability, can support changes in taxonomy of studied groups. In this sense, the study of shell morphometry during snail development can contribute to the understanding of the structural mechanisms that creates the diversity observed. DESCRIPTION: The morphological and ontogenetic pattern differences were collected among snails from four different populations, kept under the same laboratorial conditions. It was possible to distinguish characteristic shell morphometrics for snails from each population. The snails from Barra Mansa and Floriano, locations with smaller precipitation indexes presented smaller shell aperture values. The results are discussed in terms of the role of the reproductive strategy of this species as a factor determining shell shape. CONCLUSIONS: Differences in growth allometry indicated that the whole shell forming process is different among the populations, not only the final form of the adult's shell. Some allometry relationships indicated that, during the snails' development, the increase in shell width is not proportional to the increase of the width and height of the shell aperture. Thus, there is possibly an antagonism between the adoption of K-strategy and protection against desiccation. Since the spire indices of L. unilamellata morphotypes cannot be explained by physical functional aspects, the most likely explanation is the reproductive strategy of this species.

[Prevalence of intestinal helminths in three regions of Minas Gerais State]. / Prevalência de helmintos intestinais em três mesorregiões do Estado de Minas Gerais.

A parasitological assay of feces, through the Kato-Katz method, was undertaken in 18,973 schoolchildren (7 to 14 years old), at primary school level, from a public school of the State of Minas Gerais. Three mesoregions were studied: Tri ngulo Mineiro/ Alto Para ba ( 60 municipalities); northwest Minas Gerais (13) and south/south-west regions of Minas Gerais (144). Among the examined children, 15,545 (82%) were negative; 2,863 (15%) were infected with a single species of helminthes and 565 (3%) were infected by more than one species. The prevalence rates were: A. lumbricoides 10.3%; T. trichiura 4.7%; hookworm 2.9%; E. vermicularis 1.2%; H. nana 0.4% and Taenia sp 0.2%. The largest helminthes prevalence/mesoregion were for T. trichiura (24.2%) and A. lumbricoides (18.7%) in the south/south-west region; hookworm (12.1%) and Taenia sp (0.7%) in the northwest region of Minas Gerais. Intestinal helminthiasis is still regarded as a serious public health problem, including regions where the socio-economic conditions are more favorable.

Potentiality of Achatina fulica Bowdich, 1822 (Mollusca: Gastropoda) as intermediate host of the Angiostrongylus costaricensis Morera & Céspedes 1971.

Samples of Achatina fulica were experimentally infected with Angiostrongylus costaricensis larvae, etiological agent of abdominal angiostrongyliasis, showing that A. fulica is susceptible to the parasite. Achatina fulica may be a risk to urbanization of abdominal angiostrongyliasis presumably due to its high proliferation, continuous dispersion and remarkable adaptation in several Brazilian towns.