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BACKGROUND: Angiostrongylus cantonensis is the etiological agent of neuroangiostrongyliasis in humans, which is developed in gastropods and vertebrate species, mainly rodents. Human transmission occurs through consumption of molluscs and paratenic hosts infected with L3, and the migration of larvae to the central nervous system causes eosinophilic meningitis. Laboratory diagnosis is based on molecular and immunological tests, using young or adult females as a source of antigens. However, these tests give positive results only after several weeks of symptoms onset and also cross-reactions with others parasite infections may occur. OBJECTIVES: The purpose of this work was to study different antigenic preparations of distinct evolutionary phases of A. cantonensis, in order to improve serological techniques for disease immunodiagnosis. METHODS: For this purpose, antigenic fractions of different evolutionary forms were evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) and Western blot using serum samples. FINDINGS: All analysed fractions showed reactivity with serum samples from patients with neuroangiostrongyliasis, especially female membrane alkaline (FAM) and female soluble alkaline (FAS) fractions together with female soluble saline (FSS), improving the technique specificity. MAIN CONCLUSIONS: The results point to the possibility of use of raw female antigens in association with alkaline membrane antigens extracted from adult worms to aid in diagnosis and helps initiate neuroangiostrongyliasis surveillance and control actions.

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Several studies have described the effects of human tumor necrosis factor alpha (hTNF-α) on Schistosoma mansoni. hTNF-α affects the worm's development, metabolism, egg-laying, gene expression and protein phosphorylation. The available data on the influence of hTNF-α on egg-laying in S. mansoni are controversial, but understanding the mechanism of egg-laying regulation in this species is essential in combating schistosomiasis. We characterized the effects of in vitro treatment of S. mansoni adult worms with different doses of hTNF-α (5, 20 and 40 ng/ml) for 5 days. We explored the effects on egg-laying rate, glucose levels, ATP metabolism, and messenger RNA (mRNA) expression levels of lactate dehydrogenase, glucose transporters and the parasite gene which acts as an hTNF-α receptor, SmTNFR. hTNF-α influenced egg-laying in a time- and dose-dependent manner: at a dose of 40 ng/ml, egg-laying increased on day 2 and decreased on days 3 and 4; at 20 ng/ml, egg-laying decreased on day 3; while at 5 ng/ml, egg-laying decreased on day 4. The total number of eggs produced was not affected by the different treatments, but the egg-laying dynamics were: the median egg-laying time decreased significantly with treatment, and egg developmental stages and size were also affected. At 5 and 20 ng/ml hTNF-α, lactate production diminished on day 3 up to day 5, while glucose uptake increased on day 5. At 40 ng/ml, glucose uptake diminished on day 1 up to day 3, while ATP accumulation was detected on day 5. No significant changes in mRNA expression were detected in any of the treatments. We found that crosstalk involving hTNF-α and parasite signaling plays a role in the fine-scale regulation of the worm's metabolism and physiology, and points to new strategies for disease control.

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The Indirect Immunofluorescence Assay (IFA) was used to identify stage-specific antigenic structures in paraffin sections of female larvae and worms and snails tissues, infected with third stage larvae of Angiostrongylus cantonensis. Sera from eosinophilic meningitis cases were used to assess reactivity. Non-reactive sera from patients with other parasitic diseases and from individuals without other etiologies were used as controls for cross-reactivity. Larvae and worms showed high reactivity to IgG antibodies. IgM antibodies reacted with low intensity only to larvae. Fluorescent reactions were observed in the cuticles and internal structures on worms sections, with a marked reaction in the uterus content. In the snail tissues, the larvae were found exclusively inside the granulomas, with fluorescent markings in the cuticles of the larvae and inside the granulomatous tissues. This fluorescent pattern suggests the presence of excretory/secretory antigens distributed throughout the granulomas. Expressive cross-reactivity occurred in sera from patients with other parasitic diseases, especially strongyloidiasis. The use of IFA applied to paraffin sections to identify structures with antigenic potential and the study of new serological markers, can contribute to the improvement of the laboratory diagnosis of eosinophilic meningitis. (AU)

A Reação de Imunofluorescência Indireta (RIFI) foi utilizada para localizar antígenos em estruturas estágio-específicas em cortes parafinados de vermes fêmeas e em tecidos de caramujos do Gênero Biomphalaria infectados com larvas de terceiro estágio de Angiostrongylus cantonensis. Soros de casos confirmados de meningite eosinfílica foram usados para avaliação da reatividade. Soros não reagentes de casos suspeitos; de pacientes com outras parasitoses e de indivíduos sem outras etiologias foram utilizados como controle de reatividade cruzada. Anticorpos da classe IgG foram reativos para antígenos presentes nos dois estágios e, anticorpos IgM somente para o estágio larvário. Nos cortes de vermes, as marcações fluorescentes foram assinaladas nas cutículas e estruturas internas, com acentuada marcação para os conteúdos uterinos. Nos tecidos dos caramujos as larvas foram encontradas exclusivamente no interior dos granulomas, com marcações fluorescentes nas cutículas das larvas e no interior dos tecidos granulomatosos. O padrão de fluorescência no granuloma sugere a marcação de antígenos excretores/secretores. Reatividade cruzada mais expressiva ocorreu com anticorpos presentes em soros de pacientes com outras parasitoses, com destaque para estrongiloidíase. A RIFI em cortes parafinados abre novas perspectivas para identificação de antígenos e de marcadores sorológicos, que possam ser aplicados no aprimoramento do diagnóstico laboratorial da meningite eosinofílica. (AU)

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BACKGROUND Angiostrongylus cantonensis is the etiological agent of neuroangiostrongyliasis in humans, which is developed in gastropods and vertebrate species, mainly rodents. Human transmission occurs through consumption of molluscs and paratenic hosts infected with L3, and the migration of larvae to the central nervous system causes eosinophilic meningitis. Laboratory diagnosis is based on molecular and immunological tests, using young or adult females as a source of antigens. However, these tests give positive results only after several weeks of symptoms onset and also cross-reactions with others parasite infections may occur. OBJECTIVES The purpose of this work was to study different antigenic preparations of distinct evolutionary phases of A. cantonensis, in order to improve serological techniques for disease immunodiagnosis. METHODS For this purpose, antigenic fractions of different evolutionary forms were evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) and Western blot using serum samples. FINDINGS All analysed fractions showed reactivity with serum samples from patients with neuroangiostrongyliasis, especially female membrane alkaline (FAM) and female soluble alkaline (FAS) fractions together with female soluble saline (FSS), improving the technique specificity. MAIN CONCLUSIONS The results point to the possibility of use of raw female antigens in association with alkaline membrane antigens extracted from adult worms to aid in diagnosis and helps initiate neuroangiostrongyliasis surveillance and control actions.

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RESUMO

Neste estudo, Licochalcone A (LicoA) foi investigado em ensaios in vitro e in vivo . A sobrevivência dos vermes em cultura, o padrão de oviposição, a contagem de tubérculos íntegros e a integridade da pelagem foram adotados nos testes in vitro . Após a perfusão dos animais, o número de vermes recuperados, sua localização e o estudo do oograma foram os parâmetros analisados ​​para sinalizar a existência de potencial atividade esquistossomicida in vivo . Observamos redução na sobrevivência, integridade do tegumento e reprodução de vermes adultos in vitro.. Os modelos murinos não mostraram diferença significativa nos parâmetros parasitológicos analisados ​​que indicam atividade contra os vermes com uma dose oral única de 25 mg / kg de LicoA ou duas injeções intraperitoneais de 50 mg / kg de LicoA. No entanto, é muito cedo para excluir completamente a atividade esquistossomicida do LicoA, visto que a forma farmacêutica utilizada não poderia fornecer uma absorção regular do medicamento.

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Neste estudo, Licochalcone A (LicoA) foi investigado em ensaios in vitro e in vivo . A sobrevivência dos vermes em cultura, o padrão de oviposição, a contagem de tubérculos íntegros e a integridade da pelagem foram adotados nos testes in vitro . Após a perfusão dos animais, o número de vermes recuperados, sua localização e o estudo do oograma foram os parâmetros analisados ​​para sinalizar a existência de potencial atividade esquistossomicida in vivo . Observamos redução na sobrevivência, integridade do tegumento e reprodução de vermes adultos in vitro.. Os modelos murinos não mostraram diferença significativa nos parâmetros parasitológicos analisados ​​que indicam atividade contra os vermes com uma dose oral única de 25 mg / kg de LicoA ou duas injeções intraperitoneais de 50 mg / kg de LicoA. No entanto, é muito cedo para excluir completamente a atividade esquistossomicida do LicoA, visto que a forma farmacêutica utilizada não poderia fornecer uma absorção regular do medicamento.(AU)

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Angiostrongylus cantonensis, a rat lungworm, is one of the leading causes of eosinophilic meningitis in humans. Infection in humans occurs by the ingestion of intermediate hosts, undercooked paratenic hosts or contaminated vegetables and fruits by mucus from infected molluscs. This zoonosis is widespread in tropical and subtropical areas of Southeast Asia, it has also been reported in the Pacific Islands as well as in other regions of Americas. In Brazil, human cases of angiostrongyliasis have been reported since 2007 in Southeast, Northeast, and South regions. In January 2011, we collected a batch of 30 Belocaulus willibaldoi slides in a neighborhood of São Paulo city (Parque Fernanda). Six of them were used for identifying species, and the others (24) were used in parasitological tests through digestion in peptic solution and then larvae isolation by the Rugai method. A total of 250 larvae were obtained and they had morphological traits of Angiostrongylus spp. Later, four Golden hamsters (Mesocricetus auratus) were infected with 38 larvae that allowed the recovery of young worms from the brain and lungs of rodents on the 21st and 30th day of infection. In this same neighborhood we captured rodents (Rattus norvegicus) that, after necropsy led us to recovery of 22 adult worms in the pulmonary arteries (14 males and 8 females) in May 2011. The larvae and worms obtained from natural infection were evaluated by morphological and morphometric parameters, as well as biological behavior patterns and molecular profile. All methodologies identified the parasite as Angiostrongylus cantonensis. In this way, we report for the first time, the natural infection by A. cantonensis in intermediate (B. willibaldoi) and definitive (R. norvegicus) hosts in a new urban region of Brazil.

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Foi realizada reavaliação sobre o estado de preservação de ovos de helmintos e cistos de protozoários mantidos por cerca de 40 anos em solução de iodo‑mercurato de potássio a 0,2%. Foi observado que ovos de Schistosoma mansoni, Ancylostomidae e Trichuris trichiura e oocistos de Isospora belli mantiveram‑se em condições adequadas para a sua identificação ao microscópio ótico comum. No material examinado, foi possível verificar a presença de miracídio em ovo de Schistosoma mansoni, forma larvada em ovo de T. trichiura e esporozoitos em oocistos de I. belli.(AU)

A reassessment was carried out on the preservation status of helminth eggs and protozoan cysts maintained for about 40 years in 0.2% potassium iodine‑mercurate solution. It was observed that Schistosoma mansoni, Ancylostomidae and Trichiuris trichiura eggs and Isospora belli oocysts were kept in conditions suitable for their identification under a common light microscope. In the examined material, it was possible to verify the presence of miracidium in S. mansoni egg, larvae in T. trichiura egg and sporozoites in I. belli oocysts.(AU)

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Foi realizada reavaliação sobre o estado de preservação de ovos de helmintos e cistos de protozoários mantidos por cerca de 40 anos em solução de iodo­mercurato de potássio a 0,2%. Foi observado que ovos de Schistosoma mansoni, Ancylostomidae e Trichuris trichiura e oocistos de Isospora belli mantiveram­se em condições adequadas para a sua identificação ao microscópio ótico comum. No material examinado, foi possível verificar a presença de miracídio em ovo de Schistosoma mansoni, forma larvada em ovo de T. trichiura e esporozoitos em oocistos de I. belli. (AU)

A reassessment was carried out on the preservation status of helminth eggs and protozoan cysts maintained for about 40 years in 0.2% potassium iodine­mercurate solution. It was observed that Schistosoma mansoni, Ancylostomidae and Trichiuris trichiura eggs and Isospora belli oocysts were kept in conditions suitable for their identification under a common light microscope. In the examined material, it was possible to verify the presence of miracidium in S. mansoni egg, larvae in T. trichiuraegg and sporozoites in I. belli oocysts. (AU)

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