The iron distribution and magnetic properties of schistosome eggshells: implications for improved diagnostics.

BACKGROUND: Schistosoma mansoni and Schistosoma japonicum are the most frequent causative agents of human intestinal schistosomiasis. Approximately 200 million people in the world are infected with schistosomes. Diagnosis of schistosomiasis is often difficult. High percentages of low level infections are missed in routine fecal smear analysis and current diagnostic methodologies are inadequate to monitor the progress of parasite control, especially in areas with low transmission. Improved diagnostic methods are urgently needed to evaluate the success of elimination programs. Recently, a magnetic fractionation method for isolation of parasite eggs from feces was described, which uses magnetic microspheres to form parasite egg - magnetic microsphere conjugates. This approach enables screening of larger sample volumes and thus increased diagnostic sensitivity. The mechanism of formation of the conjugates remains unexplained and may either be related to specific surface characteristics of eggs and microspheres or to their magnetic properties. METHODS/PRINCIPAL FINDINGS: Here, we investigated iron localization in parasite eggs, specifically in the eggshells. We determined the magnetic properties of the eggs, studied the motion of eggs and egg-microsphere conjugates in magnetic fields and determined species specific affinity of parasite eggs to magnetic microspheres. Our study shows that iron is predominantly localized in pores in the eggshell. Parasite eggs showed distinct paramagnetic behaviour but they did not move in a magnetic field. Magnetic microspheres spontaneously bound to parasite eggs without the presence of a magnetic field. S. japonicum eggs had a significantly higher affinity to bind microspheres than S. mansoni eggs. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the interaction of magnetic microspheres and parasite eggs is unlikely to be magnetic in origin. Instead, the filamentous surface of the eggshells may be important in facilitating the binding. Modification of microsphere surface properties may therefore be a way to optimize magnetic fractionation of parasite eggs.

Ghrelin, leptin and insulin in cirrhotic children and adolescents: relationship with cirrhosis severity and nutritional status.

OBJECTIVE: Ghrelin, leptin, and insulin concentrations are involved in the control of food intake and they seem to be associated with anorexia-cachexia in cirrhotic patients. The present study aimed to investigate the relationship between the nutritional status and fasting ghrelin, leptin and insulin concentrations in pediatric cirrhotic patients. METHODS: Thirty-nine patients with cirrhosis and 39 healthy controls aged 0-15 years matched by sex and age were enrolled. Severity of liver disease was assessed by Child-Pugh classification, and Pediatric for End Stage Liver Disease (PELD) or Model for End-stage Liver Disease (MELD) scores. Blood samples were collected from patients and controls to assay total ghrelin, acyl ghrelin, leptin and insulin by using a commercial ELISA kit. Anthropometry parameters used were standard deviation score of height-for-age and triceps skinfold thickness-for-age ratio. A multiple linear regression analysis was used to determine the correlation between dependent and independent variables. RESULTS: Acyl ghrelin was significantly lower in cirrhotic patients than in controls [142 (93-278) pg/mL vs 275 (208-481) pg/mL, P=0.001]. After multiple linear regression analysis, total ghrelin and acyl ghrelin showed an inverse correlation with age; acyl ghrelin was associated with the severity of cirrhosis and des-acyl ghrelin with PELD or MELD scores ≥15. Leptin was positively correlated with gender and anthropometric parameters. Insulin was not associated with any variable. CONCLUSION: Low acyl ghrelin and high des-acyl ghrelin concentrations were associated with cirrhosis severity, whereas low leptin concentration was associated with undernourishment in children and adolescents with cirrhosis.

The effects of anesthetic regimen in 90% hepatectomy in rats / Os efeitos do regime anestésico na hepatectomia de 90% em ratos

PURPOSE: To evaluate the influence of the anesthetic regimen on anesthetic recovery, survival, and blood glucose levels following a 90% partial hepatectomy in rats. METHODS: Thirty adult male Wistar rats were divided into two groups according to their anesthetic regimens: intraperitoneal ketamine and xylazine or inhaled isoflurane. In order to prevent hypoglycemia, glucose was administered intraperitoneally and glucose (20%) was added to the drinking water. RESULTS: Anesthetic recovery time was longer in the ketamine and xylazine group. The survival rate after 72 hours was lower (log rank=0.0001) in the ketamine and xylazine group (0.0%) than in the isoflurane group (26.7%). The blood glucose after six hours was lower (p=0.017) in the ketamine and xylazine group (63±31.7 mg/dL) than in the isoflurane group (98±21.2 mg/dL). The prolonged anesthesia recovery time associated with ketamine and xylazine decreased the survival rate and blood glucose levels after 90% hepatectomy. CONCLUSION: Isoflurane anesthesia reduced the recovery time and incidence of hypoglycemia and increased the survival rate in the early hours, providing a therapeutic window that is suitable for experimental studies.

OBJETIVO: Avaliar a influência do regime anestésico sobre a recuperação anestésica, a sobrevida em 72 horas e a glicemia após hepatectomia parcial de 90% em ratos. MÉTODOS: Trinta ratos Wistar machos adultos foram distribuídos em dois grupos conforme o regime anestésico: combinação de ketamina e xilazina intraperitoneal ou isoflurano inalatório. Para prevenção de hipoglicemia foi administrada glicose intraperitoneal e adicionado glicose (20%) na água de beber. RESULTADOS: A recuperação anestésica no grupo ketamina e xilazina foi mais prolongada. Durante primeira hora após hepatectomia, nenhum rato anestesiado com ketamina e xilazina despertou. Todos do grupo isoflurano estavam ativos minutos após final da cirurgia. A sobrevida em 72 horas foi menor (Log rank=0,0001) no grupo ketamina e xilazina (0,0%) que no grupo isoflurano (26,7%). Glicemia em 6 horas do grupo ketamina e xilazina (63±31,7 mg/dL) foi menor (p=0,017) que no grupo isoflurano (98 ±21,2 mg/dL). Prolongado tempo de recuperação anestésica com ketamina e xilazina diminuiu sobrevida e glicemia após hepatectomia 90%. CONCLUSÃO: Anestesia com isoflurano reduziu tempo de recuperação e hipoglicemia, além de aumentar a sobrevida nas primeiras horas, possibilitando uma janela terapêutica adequada para estudos experimentais.

Use of monoclonal faecal elastase-1 concentration for pancreatic status assessment in cystic fibrosis patients.

OBJECTIVE: To assess the concentration of faecal elastase-1 (EL-1) in pediatric patients with cystic fibrosis with mutation DeltaF508. METHODS: Cross-sectional study with samples collected consecutively from 51 patients aged 4 months to 17 years old (mean 9.11±4.74); 32 (62.8%) patients were male. Clinical-demographic data were collected, as well as data on the type of mutation. Exocrine pancreatic insufficiency was established by the activity of faecal EL-1 < 200 µg/g. EL-1 was quantified through the monoclonal ELISA method (ScheBo Biotech AG, Germany). Pancreatic supplements were used in 46 (90.2%) patients. RESULTS: Forty-one (80.4%) patients presented with pancreatic insufficiency (EL-1 fecal < 100 µg/g): 17 (41.5%) were homozygous, 14 were heterozygous (34.1%) and 10 were non-DeltaF508 (24.4%). Regarding the mutation, there was a statistically significant association of homozygosity with faecal EL-1 concentration < 100 µg/g (p = 0.010). All patients considered to be pancreatic insufficient (n = 41) by the test were using pancreatic supplements. Ten (19.6%) presented faecal EL-1 > 200 µg/g, and 5/10 (50%) used enzymes. CONCLUSIONS: The activity of faecal EL-1 < 100 µg/g, indicating pancreatic insufficiency, was observed in 17/17 (100%) of homozygous patients, as expected, and was less frequent in patients who were heterozygous for DeltaF508 and in patients without the mutation. There was no association of faecal EL-1 concentration with age and sex of patients. The test was standardized, is easy to execute, and can be used to assess the pancreatic status of patients with cystic fibrosis.

Utilidade da concentração da elastase-1 fecal monoclonal na avaliação da função pancreática nos pacientes com fibrose cística / Use of monoclonal faecal elastase-1 concentration for pancreatic status assessment in cystic fibrosis patients

OBJETIVO: Avaliar a concentração da elastase-1 (EL-1) fecal em pacientes pediátricos com fibrose cística, portadores da mutação ∆F508. MÉTODOS: Estudo transversal com amostras colhidas consecutivamente de 51 pacientes com idade entre 4 meses e 17 anos (média 9,11±4,74), sendo 32 (62,8 por cento) pacientes do sexo masculino. Houve coleta de dados clínico-demográficos e do tipo de mutação. A insuficiência pancreática exócrina foi definida pela atividade da EL-1 fecal < 200 µg/g. A quantificação da EL-1 foi realizada pelo método ELISA monoclonal (ScheBo Biotech AG, Germany). A suplementação pancreática foi utilizada em 46 (90,2 por cento) pacientes. RESULTADOS: Quarenta e um (80,4 por cento) pacientes apresentaram insuficiência pancreática (EL-1 fecal < 100 µg/g), sendo 17 (41,5 por cento) homozigotos, 14 heterozigotos (34,1 por cento) e 10 sem ∆F508 (24,4 por cento). Ao considerar a mutação, houve associação estatisticamente significativa entre os homozigotos e a concentração da EL-1 fecal < 100 µg/g (p = 0,010). Todos os pacientes considerados insuficientes pancreáticos (n = 41) pelo teste utilizavam suplemento pancreático. Dez (19,6 por cento) apresentaram EL-1 fecal > 200 µg/g, e 5/10 (50 por cento) utilizavam enzimas. CONCLUSÕES: A atividade de EL-1 fecal < 100 µg/g, indicativa de insuficiência pancreática, apresentou-se em 17/17 (100 por cento) dos homozigotos, conforme o esperado, sendo menos frequente nos heterozigotos para ∆F508 e nos pacientes com ausência dessa mutação. Não houve relação entre a concentração da EL-1 fecal com idade e sexo dos pacientes. O teste foi padronizado, é de fácil execução e poderá ser utilizado para avaliação da função pancreática dos pacientes com fibrose cística.

OBJECTIVE: To assess the concentration of faecal elastase-1 (EL-1) in pediatric patients with cystic fibrosis with mutation ∆F508. METHODS: Cross-sectional study with samples collected consecutively from 51 patients aged 4 months to 17 years old (mean 9.11±4.74); 32 (62.8 percent) patients were male. Clinical-demographic data were collected, as well as data on the type of mutation. Exocrine pancreatic insufficiency was established by the activity of faecal EL-1 < 200 µg/g. EL-1 was quantified through the monoclonal ELISA method (ScheBo Biotech AG, Germany). Pancreatic supplements were used in 46 (90.2 percent) patients. RESULTS: Forty-one (80.4 percent) patients presented with pancreatic insufficiency (EL-1 fecal < 100 µg/g): 17 (41.5 percent) were homozygous, 14 were heterozygous (34.1 percent) and 10 were non-∆F508 (24.4 percent). Regarding the mutation, there was a statistically significant association of homozygosity with faecal EL-1 concentration < 100 µg/g (p = 0.010). All patients considered to be pancreatic insufficient (n = 41) by the test were using pancreatic supplements. Ten (19.6 percent) presented faecal EL-1 > 200 µg/g, and 5/10 (50 percent) used enzymes. CONCLUSIONS: The activity of faecal EL-1 < 100 µg/g, indicating pancreatic insufficiency, was observed in 17/17 (100 percent) of homozygous patients, as expected, and was less frequent in patients who were heterozygous for ∆F508 and in patients without the mutation. There was no association of faecal EL-1 concentration with age and sex of patients. The test was standardized, is easy to execute, and can be used to assess the pancreatic status of patients with cystic fibrosis.

Implementação de um novo modelo de experimentação animal: Zebrafish / Implementation of a new experimental animal model: Zebrafish

O Danio rerio (zebrafish, paulistinha) é um pequeno peixe de água-doce, que vem sendo utilizado como modelo de animal para o estudo de numerosas doenças humanas. A sua facilidade de manutenção e reprodução e os métodos laboratoriais para sua criação já estão bem estabelecidos. Sua manutenção requer alguns cuidados básicos e a compra de equipamentos específicos, porém estes são de baixo custo. Este peixe é considerado um bom modelo para o estudo de doenças humanas, pode servir como uma relevante plataforma para estudo de eventos moleculares, estratégias terapêuticas e avaliação dos mecanismos fisiológicos de algumas patologias. O Laboratório Experimental de Hepatologia e Gastroenterologia (LEHG), do Centro de Pesquisas do Hospital de Clínicas de Porto Alegre, iniciou em 2008 estudos na área da Hepatologia com o Zebrafish, aqui apresentados.

Danio rerio (zebrafish, paulistinha) is a freshwater small fish that has been used as an animal model for studies of several human diseases. Its characteristics of being easily bred and reproduced and its laboratory housing methods are well established. It requires some basic care and the acquisition of specific low-cost equipment. Since this fish has already been considered a good human disease model, it may serve as a powerful preclinical platform for the study of molecular events, therapeutic strategies, and for evaluating the physiological mechanisms of some pathologies. The Laboratory of Experimental Hepatology and Gastroenterology (LEHG) of the Research Center of Hospital de Clínicas de Porto Alegre initiated studies with the zebrafish in 2008, presented here.

Longitudinal clinical and serological survey of abdominal angiostrongyliasis in Guaporé, southern Brazil, from 1995 to 1999

Angiostrongilíase abdominal é uma zoonose causada pelo Angiostrongylus costaricensis, nematódeo que se localiza no interior de vasos mesentéricos. Nosso objetivo foi de abordar vários aspectos da história natural da parasitose, num estudo longitudinal clínico-sorológico. Um total de 179 indivíduos residentes em área rural no sul do Brasil, com transmissão ativa, foram seguidos por cinco anos. Neste período foram registradas prevalências de 28,2%, 4,2%, 10%, 20,2% e 2,8% e incidências de 0%, 5,9%, 8% e 1,5%. Tanto o sexo masculino quanto o feminino foram afetados com maiores frequências na faixa etária dos 30 aos 49 anos. Em 32 indivíduos, amostras de soro foram coletadas em todas as etapas e a reatividade de IgG detectada por ELISA foi variável e geralmente não persistindo mais do que um ano. Alguns padrões individuais foram sugestivos de re-infecção. Não houve associação com a ocorrência nem de dor abdominal nem com outras enteroparasitoses e não houve nenhum caso com diagnóstico confirmado (histopatológico) da infecção. Moluscos foram encontrados portando larvas infectantes de terceiro estadio, em algumas moradias, com uma prevalência geral de 16% e baixas cargas parasitárias. Em conclusão, a angiostrongilíase abdominal no sul do Brasil pode ser uma infecção frequente, porém com baixa morbidade e reatividade sorológica de gradual declínio.

The initial epidemiological studies in the low endemicity schistosomiasis area in esteio, Rio Grande do Sul, the southernmost Brazilian State, 1997 to 2000

Nor Biomphalaria glabrata neither Schistosoma mansoni were reported from Rio Grande do Sul, the southernmost Brazilian state before 1997. Their detection next to the Sinos River, Esteio, confirmed predictions of schistosomiasis expansion to the south. Parasitological examinations both in snails and fecal samples from the human population were performed from 1997 to 2000. The last 3 out of 5 surveys were performed after a preliminar serological screening procedure in a risk group identified at a population census. A total of 11 infected individuals were found infected and snails from 2 different sites were positive for S. mansoni. Samples from these 2 and other sites were identified as B. glabrata. Egg counts in feces were below 1 per gram in 6 out of 11 patients. Some socio-cultural perceptions of water contact activities next to the Sinos River may cause difficulties to control efforts, but they also may be partially acting against a very rapid increase in transmission intensity. The southernmost schistomiasis mansoni foci in Americas rise the alert for its ongoing expansion.

The initial epidemiological studies in the low endemicity schistosomiasis area in esteio, Rio Grande do Sul, the southernmost Brazilian State, 1997 to 2000

Nor Biomphalaria glabrata neither Schistosoma mansoni were reported from Rio Grande do Sul, the southernmost Brazilian state before 1997. Their detection next to the Sinos River, Esteio, confirmed predictions of schistosomiasis expansion to the south. Parasitological examinations both in snails and fecal samples from the human population were performed from 1997 to 2000. The last 3 out of 5 surveys were performed after a preliminar serological screening procedure in a risk group identified at a population census. A total of 11 infected individuals were found infected and snails from 2 different sites were positive for S. mansoni. Samples from these 2 and other sites were identified as B. glabrata. Egg counts in feces were below 1 per gram in 6 out of 11 patients. Some socio-cultural perceptions of water contact activities next to the Sinos River may cause difficulties to control efforts, but they also may be partially acting against a very rapid increase in transmission intensity. The southernmost schistomiasis mansoni foci in Americas rise the alert for its ongoing expansion.

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction-restriction fragment length polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Ovos e órgäos reprodutores de fêmeas de Angiostrongylus costaricensis säo reconhecidos mais intensamente por soros humanos de fase aguda na angiostrongilíase abdominal / Eggs and reproductive organs of female Angiostrongylus costaricensis are more intensely recognized by human sera from acute phase in abdominal angiostrongyliasis

O Angiostrongylus costaricensis é um nematódeo intra-arterial de roedores. O homem acidentalmente pode se infectar ao ingerir alimentos ou água contaminados. Nosso objetivo foi o de descrever as estruturas do parasita que säo reconhecidas por soros humanos das fases aguda e convalescente da angiostrongilíase abdominal. O método de imunofluorescência indireta foi empregado para estudar a reatividade sobre ovos íntegros e cortes de vermes fêmeas e de larvas de primeiro estágio (L1). L1 também foram estudadas íntegras e depois de tratamento por sonicaçäo. Fluorescência sempre mais intensa com soros de fase aguda foi detectada na superfície dos ovos inteiros e nos fragmentos de L1 e näo estava presente nem nas L1 inteiras, nem em seus cortes. Uma reatividade inespecífica foi detectada na borda cuticular da cavidade geral e sobre os órgäos reprodutores. Os dados indicam que estes órgäos säo fonte importante de antigenicidade

Natural infection of deroceras laeve (Mollusca: gastropoda) with Metastrongylid larvae in a transmission focus of abdominal angiostrongyliasis

Angiostrongylus costaricensis is a nematode parasitic of rodents. Man may become infected by ingestion of the third stage larvae produced within the intermediate hosts, usually slugs from the family Veronicellidae. An epidemiological study carried out in a locality in southern Brazil (western Santa Catarina State) where these slugs are a crop pest and an important vector for A. costaricensis has documented for the first time the natural infection of Deroceras laeve with metastrongylid larvae. This small limacid slug is frequently found amid the folds of vegetable leaves and may be inadvertently ingested. Therefore D. laeve may have an important role in transmission of A. costaricensis to man

Dogs may be a reservoir host for Angiostrongylus costaricensis

Angiostrongylus costaricensis is a parasitic nematode of wild rodents. Several other vertebrate species including man may become infected by ingestion of the third stage larvae produced by the intermediate hosts, usually slugs from the family Veronicellidae. There is a report of the diagnosis of abdominal angiostrongyliasis in Canis familiaris with lesions resembling those found in human disease. As a preliminar evaluation of the adequacy of a canine model for pathogenetic studies, a dog was inoculated with 75 L3 of A. costaricensis. Infection was established and fist stage larvae were found in feces up to 88 days post infection, sometimes in very large numbers (9.5 x 10(4) L1/g). No clinical manifestations or significant lesions were detected. These are indications that dog may play a role as a reservoir host for A. costaricensis