Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni.

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

[Serology as a diagnostic tool for surveillance and control of schistosomiasis in Holambra, São Paulo State, Brazil]. / Utilização de método sorológico como ferramenta diagnóstica para implementação da vigilância e controle da esquistossomose no Município de Holambra, São Paulo, Brasil.

The present study aimed to evaluate the incorporation of the immunofluorescence test (IFT) with adult parasite paraffin sections as antigen substrate for the detection of IgM antibodies (IgM-IFT), as a diagnostic method in the schistosomiasis control program in the county (municipality) of Holambra, São Paulo State, Brazil. This city was selected for this study based on its low endemicity for schistosomiasis, the first cases having been reported in 1993, and because of the need to implement a control program with more sensitive diagnostic techniques. 202 individuals underwent IgM-IFT, with 48 serologically positive cases; of these, 28 were tested with the Kato-Katz technique, using three stool samples. Schistosoma mansoni eggs were found in 14 individuals, with egg counts varying from 2.7 to 224 per gram of stool. The results indicate the potential usefulness of IgM-IFT as a screening test, subject to subsequent confirmation using a parasitological method, in low-endemic areas for schistosomiasis.

Immunoassays as an auxiliary tool for the serodiagnosis of Schistosoma mansoni infection in individuals with low intensity of egg elimination.

In an attempt to improve the screening of Schistosoma mansoni-infected individuals from a low-transmission area, we established a protocol that includes three independent coproscopy examinations and two serological assays. Three stool samples were examined using the Kato-Katz and free sedimentation methods and serum samples were tested by IgG-ELISA and IgM-immunofluorescence. Two hundred and sixty-nine individuals participated in the survey: 132 individuals (49%) showed positive serological test results. Of these, 16 (6%) had positive results in stool examination in the first sample batch. However, there were also cases with positive serological test results in spite of negative Kato-Katz stool examinations. Additional stool samples were obtained from these subjects and in this way an additional 11 egg-excretors were found. Our findings suggest that a screening method that combines antibody isotype detection and repeated parasitological stool examinations could increase the chances of detecting S. mansoni-infected patients.

Synthesis and evaluation of new oxamniquine derivatives.

Oxamniquine derivatives were synthesized and evaluated as novel schistosomicide agents. Oxamniquine (1,2,3,4-tetrahydro-2-[[(1-methylethyl)amino]methyl]-7-nitro-6-quinolinemethanol) was submitted to the Mannich reaction, using formaldehyde, paraformaldehyde and acetaldehyde as reagents, and gave three unexpected products: two of them were cyclized on the alkylamine side chain and another etherified on the aminequinolinemethanol group. The three compounds were biologically evaluated using mice infected with Schistosoma mansoni and showed promising activities, but had higher toxicities. For studies on structure-activity relationships, results demonstrate that the side alkylamine group can be modified with preserved activity, but that this modification is associated with increased toxicity.

Detection of Schistosoma mansoni antibodies in a low-endemicity area using indirect immunofluorescence and circumoval precipitin test.

Parasitological diagnostic methods for schistosomiasis lack sensitivity, especially in regions of low endemicity. The objective of this study was to determine the prevalence of Schistosoma mansoni infections by antibody detection using the indirect immunofluorescence assay (IFA-IgM) and circumoval precipitin test (COPT). Serum samples of 572 individuals were randomly selected. The IFA-IgM and COPT were used to detect anti-S. mansoni antibodies. Of the patients studied, 15.9% (N = 91) were IFA-IgM positive and 5.1% (N = 29) had COPT reactions (P < 0.001 by McNemar's test). Immunodiagnostic techniques showed higher infection prevalence than had been previously estimated. This study suggests that combined use of these diagnostic tools could be useful for the diagnosis of schistosomiasis in epidemiological studies in areas of low endemicity.

Aspectos epidemiológicos da esquistossomose em área dosudoeste de Minas Gerais, Brasil / Epidemiological aspects of schistosomiasis in area at the southwest of Minas Gerais, Brazil

Este estudo foi desenvolvido com o objetivo de contribuir com a vigilância da esquistossomose,no âmbito da Superintendência Regional de Saúde de Alfenas, MG, na mesorregião Sul/Sudoestedo estado, considerada não endêmica para a esquistossomose, ao contrário de outras áreas ao norte e nordeste do estado. Durante o ano de 2015, os escolares e migrantes de dois municípios dessa região, Arceburgo e Guaranésia, foram submetidos aos inquéritos parasitológico esorológico. No inquérito parasitológico, nenhum caso de esquistossomose foi detectado emArceburgo. Em Guaranésia, ovos de S. mansoni foram detectados entre os migrantes, com taxa de positividade de 13,6% (9/66), e em um único estudante. Sete membros da família, classificadacomo moradora de Guaranésia, mas determinada como oriunda de Timbaúba, PE quando investigada pela vigilância epidemiológica, foram também positivos para S. mansoni. No inquéritosorológico, a positividade para esquistossomose foi de 18,5% entre os migrantes de Guaranésia.Em relação às demais parasitoses, as taxas de positividade variaram de 12,5% a 32,3%. Os resultados sugerem diferenças em relação ao risco de exposição a S. mansoni e a importância da vigilância epidemiológica, mesmo em áreas não endêmicas, com foco nos migrantes, quando estes são oriundos de regiões endêmicas para esquistossomose.

This study was developed aiming at contributing to the schistosomiasis surveillance, within the scope of the Regional Health Superintendence of Alfenas, MG, in the South/Southwestmesoregion of the state, considered not endemic for schistosomiasis, unlike North and Northeast areas of the state. During the year of 2015, schoolchildren and migrants from two municipalitiesof this region, Arceburgo and Guaranésia, underwent parasitological and serological surveys.In the parasitological survey, no case of schistosomiasis was detected in Arceburgo. In Guaranésia, S. mansoni eggs were detected among the migrants, with a positivity rate of 13.6% (9/66),and in only one schoolchild. Seven members of his family, who were classified as residents of Guaranésia, but were determined as coming from Timbaúba, PE, when investigated by theepidemiological surveillance, they were also positive for S. mansoni. In the serological survey, the positivity for schistosomiasis was 18.5% among migrants from Guaranésia. Concerningthe other intestinal parasites, the positivity rates ranged from 12.5% to 32.3%. The results suggest differences in the risk of exposure to S. mansoni and the importance of epidemiologicalsurveillance, even in non-endemic areas, with a focus on migrants when they come from endemic regions for schistosomiasis.

Aspectos epidemiológicos da esquistossomose em área do sudoeste de Minas Gerais, Brasil / Epidemiological aspects of schistosomiasis in area at the southwest of Minas Gerais, Brazil

This study was developed aiming at contributing to the schistosomiasis surveillance, within the scope of the Regional Health Superintendence of Alfenas, MG, in the South/Southwest mesoregion of the state, considered not endemic for schistosomiasis, unlike North and Northeast areas of the state. During the year of 2015, schoolchildren and migrants from two municipalities of this region, Arceburgo and Guaranésia, underwent parasitological and serological surveys. In the parasitological survey, no case of schistosomiasis was detected in Arceburgo. In Guaranésia, S. mansoni eggs were detected among the migrants, with a positivity rate of 13.6% (9/66), and in only one schoolchild. Seven members of his family, who were classified as residents of Guaranésia, but were determined as coming from Timbaúba, PE, when investigated by the epidemiological surveillance, they were also positive for S. mansoni. In the serological survey, the positivity for schistosomiasis was 18.5% among migrants from Guaranésia. Concerning the other intestinal parasites, the positivity rates ranged from 12.5% to 32.3%. The results suggest differences in the risk of exposure to S. mansoni and the importance of epidemiological surveillance, even in non-endemic areas, with a focus on migrants when they come from endemic regions for schistosomiasis.

Este estudo foi desenvolvido com o objetivo de contribuir com a vigilância da esquistossomose, no âmbito da Superintendência Regional de Saúde de Alfenas, MG, na mesorregião Sul/Sudoeste do estado, considerada não endêmica para a esquistossomose, ao contrário de outras áreas ao norte e nordeste do estado. Durante o ano de 2015, os escolares e migrantes de dois municípios dessa região, Arceburgo e Guaranésia, foram submetidos aos inquéritos parasitológico e sorológico. No inquérito parasitológico, nenhum caso de esquistossomose foi detectado em Arceburgo. Em Guaranésia, ovos de S. mansoni foram detectados entre os migrantes, com taxa e positividade de 13,6% (9/66), e em um único estudante. Sete membros da família, classificada como moradora de Guaranésia, mas determinada como oriunda de Timbaúba, PE quando investigada pela vigilância epidemiológica, foram também positivos para S. mansoni. No inquérito sorológico, a positividade para esquistossomose foi de 18,5% entre os migrantes de Guaranésia. Em relação às demais parasitoses, as taxas de positividade variaram de 12,5% a 32,3%. Os resultados sugerem diferenças em relação ao risco de exposição a S. mansonie a importância da vigilância epidemiológica, mesmo em áreas não endêmicas, com foco nos migrantes, quando estes são oriundos de regiões endêmicas para esquistossomose.

Schistosomiasis mansoni in Bananal (State of São Paulo, Brazil): III. Seroepidemiological studies in the Palha District.

The Palha district, municipality of Bananal, State of São Paulo, Brazil, had 10.3% cases of Schistosoma mansoni diagnosed from 1994 to 2000 by coproscopy: about three times the municipality average. The immunofluorescent antibody test was used to assess gut-associated IgM antibody titers of samples from 452 inhabitants. It disclosed 129 (28.5%) positive cases. Subjects were classified according to age, sex, birthplace, and period of residence. Titers varied from 8 to 4,096 (geometric mean: 170.2). Seropositives were aged from 6 to 69 years (average: 24.5), 75% of them aged 34 or less, predominantly males (78 or 60.5%). Of all subjects, 65.7% were born and had been living in Bananal since; 24.2% came from neighboring municipalities and are residing in Bananal from two months to 89 years (average: 22.7 years). Further Kato-Katz coproscopy from 97 seropositives (geometric mean titer, 619) revealed S. mansoni eggs in 11 subjects (11.3%). Serology was deemed useful in screening subjects to be further investigated by coproscopy, considering that blood collection had better acceptance than supplying fecal samples. Higher than average serological titers may indicate new cases in endemic areas. Longitudinal studies associated with epidemiological investigation, including titer evolution are advised, as isolated data are difficult to interpret.

Saci-1, -2, and -3 and Perere, four novel retrotransposons with high transcriptional activities from the human parasite Schistosoma mansoni.

Using the data set of 180,000 expressed sequence tags (ESTs) of the blood fluke Schistosoma mansoni generated recently by our group, we identified three novel long-terminal-repeat (LTR)- and one novel non-LTR-expressed retrotransposon, named Saci-1, -2, and -3 and Perere, respectively. Full-length sequences were reconstructed from ESTs and have deduced open reading frames (ORFs) with several uncorrupted features, characterizing them as possible active retrotransposons of different known transposon families. Alignment of reconstructed sequences to available preliminary genome sequence data confirmed the overall structure of the transposons. The frequency of sequenced transposon transcripts in cercariae was 14% of all transcripts from that stage, twofold higher than that in schistosomula and three- to fourfold higher than that in adults, eggs, miracidia, and germ balls. We show by Southern blot analysis, by EST annotation and tallying, and by counting transposon tags from a Serial Analysis of Gene Expression library, that the four novel retrotransposons exhibit a 10- to 30-fold lower copy number in the genome and a 4- to 200-fold-higher transcriptional rate per copy than the four previously described S. mansoni retrotransposons [corrected]. Such differences lead us to hypothesize that there are two different populations of retrotransposons in S. mansoni genome, occupying different niches in its ecology. Examples of retrotransposon fragment inserts were found into the 5' and 3' untranslated regions of four different S. mansoni target gene transcripts. The data presented here suggest a role for these elements in the dynamics of this complex human parasite genome.

Utilização de método sorológico como ferramenta diagnóstica para implementação da vigilância e controle da esquistossomose no Município de Holambra, São Paulo, Brasil / Serology as a diagnostic tool for surveillance and control of schistosomiasis in Holambra, São Paulo State, Brazil

O presente estudo teve como objetivo avaliar a incorporação da reação de imunofluorescência indireta para pesquisa de anticorpos IgM (RIF-IgM) como método diagnóstico no programa de controle da esquistossomose do Município de Holambra, Estado de São Paulo, Brasil. O município foi selecionado para este estudo considerando-se a notificação dos primeiros casos de esquistossomose a partir de 1993 e a necessidade do município de implementar o programa com técnicas diagnósticas mais sensíveis, tendo em vista ser caracteristicamente área de baixa endemicidade. Duzentos e dois indivíduos foram submetidos à RIF-IgM, dos quais 48 apresentaram-se positivos, sendo 28 destes submetidos à técnica de Kato-Katz, examinando-se três amostras de fezes. Ovos de Schistosoma mansoni foram encontrados em 14, com carga parasitária variando de 2,7 a 224,0 ovos por grama de fezes. Os resultados indicam a potencialidade da RIF-IgM como método de triagem, para posterior confirmação exaustiva por método parasitológico, em áreas de baixa endemicidade.

The present study aimed to evaluate the incorporation of the immunofluorescence test (IFT) with adult parasite paraffin sections as antigen substrate for the detection of IgM antibodies (IgM-IFT), as a diagnostic method in the schistosomiasis control program in the county (municipality) of Holambra, São Paulo State, Brazil. This city was selected for this study based on its low endemicity for schistosomiasis, the first cases having been reported in 1993, and because of the need to implement a control program with more sensitive diagnostic techniques. 202 individuals underwent IgM-IFT, with 48 serologically positive cases; of these, 28 were tested with the Kato-Katz technique, using three stool samples. Schistosoma mansoni eggs were found in 14 individuals, with egg counts varying from 2.7 to 224 per gram of stool. The results indicate the potential usefulness of IgM-IFT as a screening test, subject to subsequent confirmation using a parasitological method, in low-endemic areas for schistosomiasis.

IgM antibodies to Schistosoma mansoni gut-associated antigens for the study of Schistosomiasis transmission in Ribeiräo Pires, Säo Paulo

The potential of an immunofluorescence test for detection of IgM antibodies against Schistosoma mansoni gut-associated antigens (IgM-IFT) was evaluated as a tool for studying aspects related to the schistosomiasis transmission in Ribeiräo Pires, in the metropolitan area of the capital of the State of Säo Paulo, Brazil. Children from a school with about 400 students, 6 to 18 years, were followed-up for two years. In the five surveys, carried out at 6-month intervals, from October 92 to October 94, serological (IgM-IFT) prevalence indices of 5.3 per cent, 5.8 per cent, 6.2 per cent, 2.9 per cent and 3.3 per cent were obtained. These indices were 7 to 10 times higher than the parasitological prevalence indices of 0.5 per cent, 0.5 per cent, 0.7 per cent, 0.4 per cent and 0 per cent determined by the Kato-Katz method. Seroconversion from IFT negative to positive was indicating possible newly acquired S. mansoni infection in three children. But confirmation of infection by fecal examination was possible in only one child. The IgM-IFT can constitute a valuable tool for the improvement of the vigilance program in low endemic areas for schistosomiasis, better characterizing the S. mansoni transmission in such areas.

Schistosomiasis mansoni in Bananal (State of Säo Paulo, Brazil). III. Seroepidemiological studies in the Palha District

The Palha district, municipality of Bananal, State of Säo Paulo, Brazil, had 10.3 percent cases of Schistosoma mansoni diagnosed from 1994 to 2000 by coproscopy: about three times the municipality average. The immunofluorescent antibody test was used to assess gut-associated IgM antibody titers of samples from 452 inhabitants. It disclosed 129 (28.5 percent) positive cases. Subjects were classified according to age, sex, birthplace, and period of residence. Titers varied from 8 to 4,096 (geometric mean: 170.2). Seropositives were aged from 6 to 69 years (average: 24.5), 75 percent of them aged 34 or less, predominantly males (78 or 60.5 percent). Of all subjects, 65.7 percent were born and had been living in Bananal since; 24.2 percent came from neighboring municipalities and are residing in Bananal from two months to 89 years (average: 22.7 years). Further Kato-Katz coproscopy from 97 seropositives (geometric mean titer, 619) revealed S. mansoni eggs in 11 subjects (11.3 percent). Serology was deemed useful in screening subjects to be further investigated by coproscopy, considering that blood collection had better acceptance than supplying fecal samples. Higher than average serological titers may indicate new cases in endemic areas. Longitudinal studies associated with epidemiological investigation, including titer evolution are advised, as isolated data are difficult to interpret

A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area for schistosomiasis

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (Säo Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 school children were submitted to ELISA and the data compared to the results of the parasitogical method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5 per cent, 43.0 per cent and 56.2 per cent by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Estudo de anticorpos IgM para vigilância epidemiológica da esquistossomose mansoni em área de baixa endemicidade / IgM antibodies for epidemiological vigilance of schistosomiasis mansoni in a low endemic area

O controle da esquistossomose no Estado de São Paulo iniciou-se ao final da década de 60, tendo como linhas mestras o uso de moluscidas e a de quimioterapia. Apesar da aparente redução nos níveis de infecção, o Sistema de Vigilância Epidemiológica do Estado de São Paulo tem registrado continuamente casos autóctones da doença, tendo-se observado ampliação das áreas de transmissão. Com o objetivo de buscar-se método diagnóstico mais sensível para fins epidemiológicos em áreas de baixa endemicidade, onde o exame de fezes se mostra pouco eficiente, uma técnica sorológica foi avaliada em quatro áreas consideradas endêmicas para Schistosoma mansoni (Sm) no Estado. Amostras de fezes e de sangue absorvido em papel-filtro foram coletadas de populações de quatro áreas de transmissão com diferentes perfis epidemiológicos, acompanhando-as, por um período de 2 anos, com cinco inquéritos, a intervalos semestrais. Dados de prevalência e incidência obtidos pela aplicação da reação de imunofluorescência para anticorpos IgM contra tubo digestivo de Sm (RIF-IgM) e do exame de fezes (Kato-Katz) foram analisados comparativamente nas quatro áreas estudadas com maior sensibilidade que pelo método parasitológico e detectar sazonidade em algumas das áreas, através da observação de taxas de soroconversão de RIF-IgM. Esta soroconversäo, passando de negativo para positivo, indicando provável infecção recente, foi mais frequente nos inquéritos realizados no primeiro semestre do ano (pós-veräo). A RIF-IgM demonstrou sert útil para estudos epidemiológicos da esquistossomose, podendo constituir método diagnóstico, tanto na fase aguda como crônica. (AU)

The control of schistosomiasis in the State of S"o Paulo started at the end of the sixties. Themain control measures included the application of molluscicides and chemotherapy. Despite the apparentdecrease of the infection levels, the Epidemiological Vigilance System of the State has continually recordedcases of the disease, and expansion of transmission areas has been observed. Since the stool examinationshowed to be insufficiently sensitive for epidemiological purposes in areas with low transmission, aserological technique was evaluated in four schistosomiasis endemic areas of the State with the aim offinding a more efficient diagnostic method. For a period of two years, five follow-up measures of prevalenceand incidence rates were obtained for the four areas, through the stool examination (Kato-Katz method)and detection of IgM antibodies to gut antigens by the immunofluorescence test (IgM-IFT) on filter paperblood samples. The comparative analysis of the data showed the occurrence of seasonal transmission insome of the studied areas, detected by the observation of differences in the seroconversion taxes.Seroconversion from IgM-IFT negative to positive, indicating newly acquired Schistosoma mansoniinfection, was observed more frequently in surveys carried out after summer holidays.The IgM-IFTproved to be a useful technique for epidemiological purposes in shistosomiasis, so that it can be appliedfor diagnosis of both acute and chronic forms of the disease. . (AU)