Immunoanalysis of different antigenic preparations of Angiostrongylus cantonensis for neuroangiostrongyliasis diagnosis improvement.

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.

Human tumor necrosis factor alpha affects the egg-laying dynamics and glucose metabolism of Schistosoma mansoni adult worms in vitro.

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.

Detection of antigens in paraffin sections of worms and snails infected by Angiostrongylus cantonensis by immunofluorescence reaction / Detecção de antígenos em cortes parafinados de vermes e caramujos infectados por Angiostrongylus cantonensis pela reação de imunofluorescência

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)

Immunoanalysis of different antigenic preparations of Angiostrongylus cantonensis for neuroangiostrongyliasis diagnosis improvement

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.

First record of natural infection by Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) in Belocaulus willibaldoi and Rattus norvegicus in an urban area of São Paulo city, SP, Brazil.

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.

Preservação de ovos de helmintos e cistos de protozoários em iodo-mercurato de potássio. Revisitando experimentos realizados há cerca de 40 anos / Preservation of helminth eggs and protozoan cysts in potassium iodine-mercurate. Revisiting experiments carried out about 40 years ago

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.

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.

Preservação de ovos de helmintos e cistos de protozoários em iodo­mercurato de potássio. Revisitando experimentos realizados há cerca de 40 anos / Preservation of helminth eggs and protozoan cysts in potassium iodine­mercurate. Revisiting experiments carried out about 40 years ago

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)

In vitro schistosomicidal and antiviral activities of Arctium lappa L. (Asteraceae) against Schistosoma mansoni and Herpes simplex virus-1.

Schistosomiasis and herpes diseases represent serious issues to the healthcare systems, infecting a large number of people worldwide, mainly in developing countries. Arctium lappa L. (Asteraceae), known as "bardana" and "burdock", is a medicinal plant popularly used for several purposes, including as antiseptic. In this study, we evaluated the in vitro schistosomicidal and antiherpes activities of the crude extract of A. lappa, which have not yet been described. Fruits of A. lappa L. were extracted by maceration with ethanol: H2O (96:4 v/v) in order to obtain the hydroalcoholic extract of A. lappa (AL). In vitro schistosomicidal assays were assessed against adult worms of Schistosoma mansoni, while the in vitro antiviral activity of AL was evaluated on replication of Herpes simplex virus type-1 (HSV-1). Cell viability was measured by MTT assay, using Vero cells and chemical composition of AL was determined by qualitative UPLC-ESI-QTOF-MS analysis. UPLC-ESI-QTOF-MS analysis of AL revealed the presence of dibenzylbutyrolactone lignans, such as arctiin and arctigenin. Results showed that AL was not cytotoxic to Vero cells even when tested at 400µg/mL. qPCR results indicated a significant viral load decreased for all tested concentrations of AL (400, 50, and 3.125µg/mL), which showed similar antiviral effect to acyclovir (50µg/mL) when tested at 400µg/mL. Also, AL (400, 200, and 100µg/mL) caused 100% mortality and significantly reduction on motor activity of all adult worms of S. mansoni. Confocal laser scanning microscopy showed tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a dose-dependent manner after treatment with AL. This report provides the first evidence for the in vitro schistosomicidal and antiherpes activities of AL, opening the route to further schistosomicidal and antiviral studies with AL and their compounds, especially lignans.

Synergistic effects of in vitro combinations of piplartine, epiisopiloturine and praziquantel against Schistosoma mansoni.

Schistosomiasis is a world health problem, and praziquantel is the only drug currently used for the treatment. There is some evidence that extensive monotherapy of praziquantel may be leading to drug resistance in the parasite. In order to find alternative treatments, the effects of the combination of epiisopiloturine (EPI), piplartine (PPT) and praziquantel (PZQ) were evaluated. Similarity analysis of these compounds was performed using optimized molecular structures to compare the shape and the charge modeling of combinations between PZQ and EPI or PPT. Supported by this data, in vitro association of PZQ-PPT, PZQ-EPI, and EPI-PPT was carried out, and the activity of these combinations against Schistosoma mansoni was assessed. The results showed synergistic activity with a combination index (CI) of 0.42 for the treatment with PZQ-PPT. Both PZQ-EPI and EPI-PPT combinations also showed synergistic effects, with CI values of 0.86 and 0.61, respectively. Surface alterations in the tegument of adult schistosomes after the treatments were observed using laser confocal microscopy and scanning electron microscopy. Additionally, the association of EPI-PPT decreased the cytotoxicity when compared with both isolated compounds in three different lines of mammalian cells. Thus, synergistic combinations of PZQ-PPT, PZQ-EPI, and EPI-PPT create the possibility of reduced doses to be used against Schistosoma mansoni.

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.

Synergistic effects of in vitro combinations of piplartine, epiisopiloturine and praziquantel against Schistosoma mansoni

Schistosomiasis is a world health problem, and praziquantel is the only drug currently used for the treatment. There is some evidence that extensive monotherapy of praziquantel may be leading to drug resistance in the parasite. In order to find alternative treatments, the effects of the combination of epiisopiloturine (EPI), piplartine (PPT) and praziquantel (PZQ) were evaluated. Similarity analysis of these compounds was performed using optimized molecular structures to compare the shape and the charge modeling of combinations between PZQ and EPI or PPT. Supported by this data, in vitro association of PZQ-PPT, PZQ-EPI, and EPI-PPT was carried out, and the activity of these combinations against Schistosoma mansoni was assessed. The results showed synergistic activity with a combination index (CI) of 0.42 for the treatment with PZQ-PPT. Both PZQ-EPI and EPI-PPT combinations also showed synergistic effects, with CI values of 0.86 and 0.61, respectively. Surface alterations in the tegument of adult schistosomes after the treatments were observed using laser confocal microscopy and scanning electron microscopy. Additionally, the association of EPI-PPT decreased the cytotoxicity when compared with both isolated compounds in three different lines of mammalian cells. Thus, synergistic combinations of PZQ-PPT, PZQ-EPI, and EPI-PPT create the possibility of reduced doses to be used against Schistosoma mansoni.

Potential for shistosomiasis in a municipality of Rondônia, Brazilian Amazon / Potencial de transmissão de esquistossomose em município de Rondônia, Amazônia brasileira

Schistossomiasis is a parasitic disease, caused by helminths of the genus Schistosoma and transmitted in Brazil by snails of the genus Biomphalaria. The municipality of Ouro Preto do Oeste, Rondônia, in the Brazilian Amazon Region, has unusually registered more than 900 cases of schistosomiasis in the last 10 years. The aim of this study was to investigate de potential of transmission of schsitosomiasis in Ouro Preto do Oeste. A total of 1,196 people in a risk area for the disease transmission were requested to answer a clinical-epidemiological survey and to collect feces samples for examination. All the samples that underwent examination resulted negative for S. mansoni. Two hundred and sixty-eight snails were collected in the locality of Ouro Preto do Oeste in 32 different locations. Among these, 44% were classified as belonging to the genus Biomphalaria. Another sample of snails (146 specimens), collected at the same sites, were submitted to an in vitro challenge with Schistosoma mansoni, and none of them were able to transmit the parasite. Finally, we discuss the epidemiological importance of these findings and the lack of attention to a patient with the disease in a non-endemic area. We failed to detected any association between shistossomiasis and the snails from the genus Biomphalaria, that exists in the local, as the planorbids were unable to transmit Shistosoma mansoni. Perhaps the small sample and/or the stool examination technique can have contributed to the results. Further studies, in other localities of Rondônia and with a greater sample could put some light in this question.

A esquistossomose é uma doença parasitária, causada por helmintos do gênero Schistosoma e transmitida no Brasil por caramujos do gênero Biomphalaria. O município de Ouro Preto do Oeste, Rondônia, Amazônia brasileira, notificou, de forma não usual, mais de 900 casos de esquistossomose mansônica nos últimos 10 anos. O objetivo deste estudo foi investigar o potencial de transmissão da esquistossomose em Ouro Preto do Oeste. Um total de 1.196 pessoas residindo em áreas de risco para a transmissão da doença foram abordadas para responderem um questionário clínico-epidemiológico e realização de exame parasitológico de fezes. Todas as amostras foram negativas para S. mansoni. Duzentos e sessenta e oito caramujos foram coletados em Ouro Preto do Oeste, de 32 localidades diferentes. Entre estes, 44% foram classificados como pertencendo ao gênero Biomphalaria. Outra amostra de caramujos (146 exemplares), coletada nos mesmos locais, foi submetida in vitro a cepas de Schistosoma mansoni, sendo todos incapazes de transmitirem o parasito. Por fim, discute-se a importância epidemiológica desses achados e a falta de atenção ao paciente portador da parasitose em áreas indenes. O estudo não conseguiu demonstrar a ocorrência de transmissão na localidade e nem a habilidade dos planorbídeos locais transmitirem Shistosoma mansoni. Talvez o pequeno tamanho da amostra e/ou a técnica de exame de fezes (pouco sensível) possam ter contribuído para este resultado. Estudos futuros, em outras localidades e com uma amostra maior podem colocar um pouco de luz nesta questão.

MOLECULAR IDENTIFICATION OF Pseudoterranova azarasi LARVAE IN COD (Gadus sp.) SOLD FOR HUMAN CONSUMPTION IN BRAZIL / Identificação molecular de larva de Pseudoterranova azarasi em bacalhau (Gadus sp.) vendido para consumo humano no Brasil

Anisakiasis and Pseudoterranovosis are human diseases caused by the ingestion of live Anisakidae larvae in raw, undercooked or lightly marinated fish. Larvae were collected from one salted cod sold for human consumption in a Sao Paulo market in 2013. One section of one brownish larva was used for molecular analyses. The partial COX2 gene sequence from the larva had a nucleotide identity of 99.8 % with Pseudoterranova azarasi, which belongs to the Pseudoterranova decipiens species complex. The risk of allergy when consuming dead larvae in salted fish is not well known and should be considered.

Os termos Anisakiasis e Pseudoterranovosis são utilizados para doença em humanos causada pela ingestão de larvas vivas de parasitas da Família Anisakidae em peixes crus, mal cozidos ou levemente marinados. As larvas foram coletadas de bacalhau salgado vendido para consumo humano num mercado de São Paulo em 2013. Uma parte da larva de cor castanha foi utilizada em análises moleculares. A sequencia parcial do gene COX2 obtida da larva mostrou 99,8% de identidade de nucleotídeos com Pseudoterranova azarasi, que faz parte do complexo de espécies Pseudoterranova decipiens. O risco de reação alérgica envolvido no consumo de larvas mortas em peixe salgado não é bem conhecido e deve ser considerado.

Comparative Study of the Accuracy of Different Techniques for the Laboratory Diagnosis of Schistosomiasis Mansoni in Areas of Low Endemicity in Barra Mansa City, Rio de Janeiro State, Brazil.

Schistosomiasis constitutes a major public health problem, with an estimated 200 million people infected worldwide. Many areas of Brazil show low endemicity of schistosomiasis, and the current standard parasitological techniques are not sufficiently sensitive to detect the low-level helminth infections common in areas of low endemicity (ALEs). This study compared the Kato-Katz (KK); Hoffman, Pons, and Janer (HH); enzyme-linked immunosorbent assay- (ELISA-) IgG and ELISA-IgM; indirect immunofluorescence technique (IFT-IgM); and qPCR techniques for schistosomiasis detection in serum and fecal samples, using the circumoval precipitin test (COPT) as reference. An epidemiological survey was conducted in a randomized sample of residents from five neighborhoods of Barra Mansa, RJ, with 610 fecal and 612 serum samples. ELISA-IgM (21.4%) showed the highest positivity and HH and KK techniques were the least sensitive (0.8%). All techniques except qPCR-serum showed high accuracy (82-95.5%), differed significantly from COPT in positivity (P < 0.05), and showed poor agreement with COPT. Medium agreement was seen with ELISA-IgG (Kappa = 0.377) and IFA (Kappa = 0.347). Parasitological techniques showed much lower positivity rates than those by other techniques. We suggest the possibility of using a combination of laboratory tools for the diagnosis of schistosomiasis in ALEs.

MOLECULAR IDENTIFICATION OF Pseudoterranova azarasi LARVAE IN COD (Gadus sp.) SOLD FOR HUMAN CONSUMPTION IN BRAZIL.

Anisakiasis and Pseudoterranovosis are human diseases caused by the ingestion of live Anisakidae larvae in raw, undercooked or lightly marinated fish. Larvae were collected from one salted cod sold for human consumption in a Sao Paulo market in 2013. One section of one brownish larva was used for molecular analyses. The partial COX2 gene sequence from the larva had a nucleotide identity of 99.8 % with Pseudoterranova azarasi, which belongs to the Pseudoterranova decipiens species complex. The risk of allergy when consuming dead larvae in salted fish is not well known and should be considered.

Evaluation of real-time PCR assay to detect Schistosoma mansoni infections in a low endemic setting.

BACKGROUND: Schistosomiasis constitutes a major public health problem, and 200 million people are estimated to be infected with schistosomiasis worldwide. In Brazil, schistosomiasis has been reported in 19 states, showing areas of high and medium endemicity and a wide range of areas of low endemicity (ALE). Barra Mansa in Rio de Janeiro state has an estimated prevalence of 1%. ALE represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with a low parasite load (<100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. The objective of this study was to evaluate the performance of a TaqMan quantitative polymerase chain reaction (qPCR) technique in serum and feces DNA samples using the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH) as references, during an epidemiological survey using fecal samples and sera from randomized residents from an ALE. METHODS: A cross-sectional study conducted from April to December 2011 using a probabilistic sampling that collected 572 fecal and serum samples. The laboratory diagnostic techniques used were: KK, HH and qPCR (feces and serum). RESULTS: We obtained the following results using the different diagnostic techniques: KK and HH, 0.9% (n =5); qPCR-feces, 9.6% (n =55); and qPCR-serum, 1.4% (n =8). The qPCR-feces presented the highest positivity, whereas the techniques of HH and KK were the least sensitive to detect infections (0.8%). Compared to HH and KK, qPCR-feces showed a statistically significant difference in positivity (p <0.05), although with poor agreement. CONCLUSION: The positivity rate presented by the qPCR approach was far higher than that obtained by parasitological techniques. The lack of adequate surveillance in ALE of schistosomiasis indicates a high possibility of these areas being actually of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using combined laboratory tools in the diagnosis of schistosomiasis in ALE.

Detection of Schistosoma mansoni infection by TaqMan® Real-Time PCR in a hamster model.

An experimental study in hamsters was performed to evaluate the capability for detecting Schistosoma mansoni DNA in serum and fecal samples during the pre and post-egg-laying periods of infection using TaqMan® Real-Time PCR system (qPCR), was compared with the circumoval precipitin test (COPT) and the Kato-Katz technique, especially among individuals with low parasitic burden. Twenty-four hamsters were infected with cercariae. Three hamsters were sacrificed per week under anesthesia, from 7 days post infection (DPI) up to 56 DPI. A serum sample and a pool of feces were collected from each hamster. The presence of S. mansoni eggs in fecal samples was evaluated by Kato-Katz method and in the hamsters gutby histopathology. Detection of S. mansoni DNA was performed using qPCR and S. mansoni antibody using COPT. The first detection of eggs in feces by Kato-Katz method and S. mansoni DNA in feces by qPCR occurred 49 DPI. Nevertheless, S. mansoni DNA was detected in serum samples from 14 up to 56 DPI. COPT was positive at 35 DPI. The results not only confirm the reliability of S. mansoni DNA detection by qPCR, but also demonstrate that serum is a trustworthy source of DNA in the pre patent infection period.

Anthelmintic activity of crude extract and essential oil of Tanacetum vulgare (Asteraceae) against adult worms of Schistosoma mansoni.

Schistosomiasis, a parasitic disease caused by trematode flatworms of the genus Schistosoma, affects more than 200 million people worldwide, and its control is dependent on a single drug, praziquantel. Tanacetum vulgare (Asteraceae) is used in folk medicine as a vermifuge. This study aimed to investigate the in vitro schistosomicidal activity of the crude extract (TV) and the essential oil (TV-EO) from the aerial parts of T. vulgare. TV-EO was obtained by hydrodistillation and analyzed by GC/MS, which allowed the identification of ß-thujone (84.13%) as the major constituent. TV and TV-EO, at 200 µg/mL, decreased motor activity and caused 100% mortality of all adult worms. At 100 and 50 µg/mL, only TV caused death of all adult worms, while TV-EO was inactive. TV (200 µg/mL) was also able to reduce viability and decrease production of developed eggs. Confocal laser scanning microscopy showed morphological alterations in the tegument of the S. mansoni surface after incubation with TV (50 and 100 µg/mL). Quantitative analysis on the schistosomes tegument showed that TV caused changes in the numbers of tubercles of S. mansoni male worms in a dose-dependent manner. The findings suggest that T. vulgare is a potential source of schistosomicidal compounds.