Abstract INTRODUCTION: In most Strongyloides stercoralis infected individuals, nematoidosis occurs asymptomatically, but in immunocompromised patients, it can cause hyperinfection. Serological techniques seem to be a good alternative for detecting this parasite. METHODS The frequency of seropositivity for strongyloidiasis in Alfenas, MG, was estimated using the enzyme linked immunosorbent assay on blood samples, between May and August of 2015. RESULTS: Out of 258 samples tested, 53.9% were positive, and the frequency of seropositive individuals was higher in the peripheral districts of the municipality. CONCLUSIONS: The results indicate high seropositivity rates for strongyloidiasis among the residents of Alfenas city.

SUMMARY Visceral leishmaniasis is a systemic and chronic disease and dogs are the main reservoir of the etiologic agent, Leishmania infantum (syn L. chagasi). A serological and molecular investigation of canine visceral leishmaniasis (CVL) was performed in the municipality of Alfenas, located in the southern region of Minas Gerais, where the disease is not endemic. Samples from 87 dogs were submitted to serological tests including the Dual Path Platform (DPP (r) ) CVL Bio-Manguinhos rapid test, an in-house enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT), as well as molecular techniques such as a conventional polymerase chain reaction (PCR) with the RV1/RV2 primers and a quantitative PCR (qPCR) with the LinJ31, Ldon and DNApol primers. Of the 87 serum samples, eight (9.2%) were positive for Leishmania using the DPP rapid test, but only four (4.6%) were confirmed by ELISA and two (2.3%) by IFAT. In these two serologically confirmed cases, spleen and liver samples were positive by all the employed molecular and parasitological procedures performed on spleen samples. When whole blood samples were used in the molecular assays, two samples (2.3%) were positive only by qPCR. DNA extracted and amplified from the spleens of seropositive dogs was sequenced, showing 100% of similarity with the Leishmania infantum (syn L. chagasi) sequence. Thus, the first cases of CVL have been confirmed in the Alfenas region, suggesting the importance of canine surveys in non-endemic municipalities for CVL to monitor disease progression and to prevent outbreaks.

Isolados de Cryptosporidium identificados em quatorze amostras de fezes, coletadas de cinco pacientes com infecção por HIV e de nove crianças imunocompetentes, residentes no estado de São Paulo, Brasil, foram submetidos a análise molecular por Nested-PCR, seguido da caracterização genética por polimorfismo do tamanho do fragmento de restrição (RFLP). A análise foi baseada na digestão, com a enzima de restrição RsaI, de um fragmento de DNA amplificado do gene que codifica a proteína de parede do oocisto de Cryptosporidium (COWP). Baseado nesta análise, quando comparadas aos padrões utilizados, quatro amostras foram identificadas como Cryptosporidium parvum, oito como Cryptosporidium hominis e duas apresentaram um perfil correspondente ao de Cryptosporidium meleagridis. O uso de métodos moleculares pode ser útil para identificar a fonte das infecções e os fatores de risco relacionados à infecção por Cryptosporidium em nossas comunidades.

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

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.

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.

Considerando a escassez de antígenos quimicamente definidos, realmente úteis e confiáveis para aplicação na soroepidemiologia da esquistossomose em larga escala, foi proposto, neste trabalho, um método alternativo para a seleção de clones de cDNA que expressam proteínas com putativo potencial diagnóstico na esquistossomose. Empregando anticorpos específicos contra uma fração proteica de 31/32 kDa (Sm31/32), purificados através da dissociação de imunocomplexos, foram selecionados cinco clones de cDNA a partir de genoteca de verme adulto de Schistosoma mansoni. O seqüenciamento parcial destes clones demonstrou que todos eram relacionados ao S. mansoni: dois apresentaram homologia com a proteína de choque térmico de 70 kDa e os demais com glutationa S-transferase, "homeodomain protein" e uma etiqueta de seqüência expressa (EST). Este último foi o clone que melhor reagiu, durante o processo de seleção, com os anticorpos anti-Sm31/32 dissociados de imunocomplexos. Baseado na seqüência de aminoácidos deste clone, dois peptídeos foram quimicamente sintetizados e analisados separadamente frente a misturas de soros de indivíduos normais e de pacientes com esquistossomose mansoni. Ambos os peptídeos demonstraram uma intensa reatividade somente contra a mistura de soros positivos, sugerindo que estes peptídeos podem ser úteis como antígenos para o diagnóstico da esquistossomose mansoni.

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

The immunoreactivity of seven peptides synthesized from Schistosoma mansoni proteins, was evaluated by dot-blot and ELISA assays using two different sensitization methodologies. The best results were obtained on wells of the Costar 3590 microplates coated with peptides P1, P2, P3, P6, and P7 using conventional methodology. The signals increased considerably (p < 0.0003) on wells sensitized with P1 to P6 using alternative methodology. In contrast, the well coated with peptide P7 presented lower signal when compared with conventional methodology (p = 0.0019). These results, establish the basis for the application of synthetic peptides for laboratory diagnosis of schistosomiasis mansoni.

IgM-ELISA is an immunoenzymatic method useful for detection of IgM antibodies against a fraction of Schistosoma mansoni adult worm antigen (AWA) that is soluble in trichloroacetic acid (AWA-TCA). This method was applied to three groups of individuals with different clinical and epidemiological characteristics, and the results compared with those obtained by other diagnostic methods: immunofluorescence test for detection of IgM antibodies (IgM-IFT) or IgG antibodies (IgG-IFT), ELISA for detection of IgG antibodies (IgG-ELISA), and two parasitological methods, Kato-Katz and miracidium hatching. The IgM-ELISA presented a sensitivity of 98%, when the parasitologic fecal examination was defined as reference diagnostic method, and a specificity of 98 and 97.3%, respectively for the group of clinically healthy individuals and other helminth carriers. A comparative analysis between the results of IgM-ELISA and those obtained by other serologic tests showed a good degree of agreement, with Kappa indices ranging from 0.95 to 0.98. The diagnostic efficacy of 97.8%, as determined with schistosomiasis patients with low parasitic burden, suggests the excellent performance of the IgM-ELISA and its usefulness for the diagnosis of schistosomiasis when applied in low endemic areas.

A field survey on schistosomiais was carried out in 1998, in the municipality of Pedro de Toledo, a low endemic area in the state of São Paulo, Brazil. According to the parasitologic Kato-Katz method, the prevalence rate was 1.6%, with an infection intensity of 40.9 eggs per gram of stool. By the immunofluorescence test (IFT) for detection of IgG and IgM antibodies in the serum, IgG-IFT and IgM-IFT, respectively, prevalence indices of 33.2% and 33.5% were observed. To assess the impact of the schistosomiasis control program in the area, parasitologic and serologic data obtained in 1998, analyzed according to the age, sex, and residence zone, were compared to previous data obtained in a epidemiologic study carried out in 1980, when prevalence indices were of 22.8% and 55.5%, respectively by Kato-Katz and IgG-IFT. A significant fall of the prevalence was observed, indicating that the control measures were effective. Nonetheless, residual transmission was observed, demonstrating the need for a joint effort to include new approaches for better understanding the real situation and improving the control of the disease in low endemic areas.

A técnica coproparasitológica de concentração em formol acetato de etila foi empregada para a quantificação de ovos de helmintos. O método quantitativo proposto foi padronizado utilizando-se o sistema comercial Coprotest® e amostras fecais contendo diferentes cargas de ovos de Ascaris lumbricoides. Para a comparação do Coprotest® quantitativo com outros métodos de quantificação de ovos, foi preparada em laboratório uma série de amostras fecais, com carga decrescente de ovos de A. lumbricoides, Trichuris trichiura e Schistosoma mansoni. Discutem-se as vantagens de se empregar um método capaz de detectar maior número de espécies de helmintos, além de protozoários, e que permita, concomitantemente, estimar a intensidade das infecções por geo-helmintos e S. mansoni nas populações. O Coprotest® quantitativo mostrou ser de aplicação viável, fornecendo resultados comparáveis a outros métodos quantitativos já descritos na literatura.

The formol ethyl acetate concentration technique was applied for the quantification of helminth eggs in fecal samples. The proposed quantitative method was standardized through the use of a commercial kit, Coprotest®, and fecal samples with different counts of Ascaris lumbricoides eggs. For the comparison of the quantitative Coprotest® with other methods of egg quantification, a series of fecal samples was prepared in laboratory, with decreasing number of A. lumbricoides, Trichuris trichiura and Schistosoma mansoni eggs. It is discussed the advantages of a method that is able of detecting different helminth and also protozoa species, allowing, in concomitance, to estimate in the populations the intensity of S. mansoni and geohelminth infections. The quantitative Coprotest® showed to be feasible, providing results that were comparable to the other quantitative methods already described in the literature.

The high sensitivity and the ability to diagnose schistosomiasis in a very early phase after infection have indicated the detection of IgM antibodies to Schistosoma mansoni gut antigens by the immunofluorescence test (IgM-IFT) as a useful serological test for epidemiological studies in low endemic areas. When applied in a follow-up study for two years, higher rates of seroconversion from IFT negative to positive were observed during the summer months, suggesting seasonal transmission of schistosomiasis in the rural area of the municipality of Itariri (São Paulo, Brazil). In each survey, blood samples from about 600 schoolchildren were collected on filter paper and submitted to IgM-IFT. When the blood samples were classified for the IgM antibody levels, according to the intensity of fluorescent reaction observed at fluorescence microscopy, and correlated to the egg counts in the Kato-Katz positive patients, no association was observed. This observation might suggest that the intensity of fluorescence observed in the IgM-IFT, as an indicator of IgM antibody levels, could not be an useful seroepidemiological marker for classifying areas of low endemicity according to degrees of infection.

O potencial diagnóstico dos anticorpos IgM e IgA contra antígenos do tubo digestivo do Schistosoma mansoni, detectados através da reação de imunofluorescência indireta utilizando-se cortes parafinados de vermes adultos, foi avaliado, comparativamente aos resultados do exame parasitológico de fezes, para fins epidemiológicos em áreas de baixa endemicidade para a esquistossomose. Amostras de sangue em papel de filtro foram coletadas de escolares, residindo em duas localidades diferentes dentro do município de Itariri (São Paulo, Brasil), com características epidemio-lógicas distintas em relação à esquistossomose. Os dados parasitológicos e sorológicos foram comparados aos obtidos com um outro grupo de escolares, residentes em uma área não endêmica para esquistossomose. Os resultados demonstraram a falta de sensibilidade do método parasitológico para detecção de indivíduos com baixa carga parasitária e indicam a potencialidade do método sorológico como importante instrumento a ser incorporado aos programas de controle e de vigilância da esquistossomose, para verificação da real situação da esquistossomose em áreas de baixa endemicidade.

The diagnostic potential of circulating IgM and IgA antibodies against Schistosoma mansoni gut-associated antigens detected by the immunofluorescence test (IFT) on adult worm paraffin sections was evaluated comparatively to the fecal parasitological method, for epidemiological purposes in low endemic areas for schistosomiasis. Blood samples were collected on filter paper from two groups of schoolchildren living in two different localities of the municipality of Itariri (São Paulo, Brazil) with different histories and prevalences of schistosomiasis. The parasitological and serological data were compared to those obtained for another group of schoolchildren from a non-endemic area for schistosomiasis. The results showed poor sensitivity of the parasitological method in detecting individuals with low worm burden and indicate the potential of the serological method as an important tool to be incorporated into schistosomiasis control and vigilance programs for determining the real situation of schistosomiasis in low endemic areas.

Uma biblioteca de expressão de cDNA de verme adulto de S. mansoni foi selecionada utilizando-se soros de macacos babuínos em fase inicial da infecção. Os clones que reagiram positivamente com os soros de infecção recente foram examinados quanto à reatividade contra soros normais e soros de infecção heteróloga. Com a finalidade de se conseguir melhor discriminação entre reatividade positiva com o anticorpo específico e aquela devido aos anticorpos anti-E. coli residuais, um clone de cDNA não relacionado ao S. mansoni foi plaqueado em mistura com o clone positivo. O plano de fundo negativo proporcionado pelo clone não relacionado forneceu o contraste necessário para discriminar a reação positiva específica. Esta metodologia de caracterização dos clones de expressão permite alcançar rapidamente os clones que apresentam o perfil de reatividade desejada, para posterior sequenciamento, subclonagem e expressão

A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

Em pacientes com esquistossomose, são encontrados anticorpos contra grande número de antígenos parasitários, e aqueles contra formas evolutivas jovens do parasita demonstraram que eram eficientes marcadores imunológicos para o diagnóstico da esquistossomose. Padrões de queda de anticorpos IgM e IgG contra cercária e esquistossômulo foram aqui estudados, comparativamente aos dos anticorpos contra verme e ovo, em pacientes esquistossomóticos após quimioterapia, abordando aspectos soroepidemiológicos. Dados obtidos no estudo de 359 amostras de soros, pertencentes a pacientes infectados por Schistosoma mansoni, indivíduos não infectados e pacientes acompanhados pós-tratamento por um período de 12 a 15 meses, forneceram base para postular padrão geral da cinética de queda de anticorpos. Antes do tratamento, o padrão de anticorpos é representado por curva unimodal, que se modifica para curva bimodal após tratamento, e finaliza com curva unimodal semelhante à do grupo de indivíduos não infectados. Diferentes tipos de anticorpos foram classificados em quatro categorias, conforme características apresentadas na queda, e o anticorpo IgM anti-esquistossômulo foi considerado como pertencente à categoria de anticorpos com queda moderada, enquanto que os demais anticorpos contra formas evolutivas jovens do parasita, à categoria daqueles com queda lenta. A metodologia aqui empregada permite identificar anticorpos potencialmente mais adequados a serem detectados em programas para controle da esquistossomose ou de outras parasitoses no campo

Antibodies to a number of parasite antigens are found in schistosomiasis patients, and antibodies to early developmental stages were demonstrated to be efficient immunologic markers for the diagnosis of schistosomiasis. In the present study, decay patterns of IgM and IgG antibodies against cercariae and schistosomula were investigated, in comparison to antibodies against worms and eggs in schistosomiasis patients after chemotherapy, for an investigation of seroepidemiologic aspects. Data obtained in the study of 359 serum samples from patients with Schistosoma mansoni infection, noninfected individuals, and patients followed-up for a period of 12 to 15 months after treatment provided the basis to postulate a general pattern for the kinetics of antibody decay. Before treatment, the antibody pattern was represented by a unimodal curve, which shifted to a bimodal curve after treatment, and ended with a unimodal curve similar to that for the noninfected group. Different types of antibodies were classified into four categories according to their decay features, and anti-schistosomulum IgM was classified into the moderate-decay caterogy, whereas other antibodies to early parasite stages were classified into the slow-decay category. The present methodology permits the identification of the most suitable antibodies to be detected in field control programs for schistosomiasis or other parasitoses

Desde 1980, está-se estudando a epidemiologia e o controle da esquistossomose mansônica no Município de Pedro de Toledo (Estado de São Paulo, Brasil). Em 1980 a prevalência avaliada por exame de fezes (método de Kato-Katz) foi de 22,8%. Estatisticamente, ao nível de 5%, não houve diferença nas prêvalencias observadas nas zonas rural e urbana. A intensidade de infecção foi baixa (média geométrica de 58,5 ovos por grama de fezes). As maiores prevalências e intensidades de infecção foram registradas na faixa etária de 5 a 29 anos. Geralmente a transmissão da endemia verificou-se durante o lazer. Apenas 0,4% de B. tenagophila mostraram-se positivos para cercárias de S. mansoni. A maioria dos portadores era assintomático. O programa de controle foi intensificado após avaliação dos dados de 1980, resultando em diminuição acentuada da prevalência de 22,8% em 1980 para 6%. Esta prevalência residual vem se mantendo até 1987. Agora iniciamos estudos para investigar as possíveis causas dessa prevalência residual.

The epidemiology and control of schistosomiasis mansoni in the Municipality of Pedro de Toledo (State of S. Paulo, Brazil) since 1980, has been studied. In 1980 the prevalence evaluated by stool exams (Kato-Katz method) was 22.8% and no statistical difference at 5.0% level was observed between rural and urban zones. The intensity of infection was low (58.5 eggs/g of faeces); the highest prevalence and intensity of infection rates were observed within the group of from 5 to 29 years of age, respectively. The transmission of schistosomiasis usually occurred during leisure time. The majority of the carriers of the parasite were asymptomatic. Of the B. tenagophila examined only 0.4% were found to be infected. The control programme has been intensified from 1981 on resulting in a sharp decrease in the prevalence from 22.8% in 1980 to 6% at the present time. This result shows that, in spite of the control programme there is a residual human prevalence. A beginning has been made on the investigation into the possible causes of this residual prevalence (6.0% was maintained through out 1987).