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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.

[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., This work was funded by grants from the Australian Research Council (ARC, DP1093471), the National Health and Medical Research Council of Australia (NHMRC, APP1002898), a University of Western Australia/University of Queensland Bilateral Research Collaboration Award, as well as grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 301979/2010-3) and the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, 09/0096-7) in Brazil. Travel to Australia by RLM and RRFC was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, 032/2010) and FAPERGS (12/1137-8 APE) grants awarded to CGT.

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.

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.

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%) 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%) pacientes. RESULTADOS: Quarenta e um (80,4%) pacientes apresentaram insuficiência pancreática (EL-1 fecal < 100 µg/g), sendo 17 (41,5%) homozigotos, 14 heterozigotos (34,1%) e 10 sem ∆F508 (24,4%). 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%) apresentaram EL-1 fecal > 200 µg/g, e 5/10 (50%) utilizavam enzimas. CONCLUSÕES: A atividade de EL-1 fecal < 100 µg/g, indicativa de insuficiência pancreática, apresentou-se em 17/17 (100%) 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%) 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-∆F508 (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 ∆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.

The Baermann method is a classical method for isolation of nematode larvae from either biological material or sou. The inclusion of a second gauze filter in the funnel resulted in a cleaner suspension of Angioslrongylus costaricensis first stage larvae (Li) from expenmentally infected rodent feces, without retention of LI as compared to the original method, if the preparation is left at rest for 12 hours. The second filter is a 2x2 cm gauze (4 layers) inserted in the opening of the straight portion of the funnel. A cleaner larvae suspension is important as a first step for purification and decontamination of the parasites to be cultivated in vitro.

Angiostrongylus costaricensis is a nematode parasite of wild rodents. In a previous experiment, it was demonstrated that bleach water (1.5%) eliminates the infective third stage larvae that may be present on vegetables and fruits. In that experiment, sem from mice moculated with inactivated larvae had humoral reactivity against crude antigen ofA. costaricensis detected by Westem-blot. In order to confirm and analyse this flnding, we used 3 groups of Swiss females, 8 weeks-old mice: a negative control group (NC), a positive control group (PC) and an experimental group (EG). Mice ofEG group were inoculatedper os with inactivated larvae. Afler 28 days, the mice ofPC and EG groups were moculated with 15 non-treated infective third stage larvae. No significant differences favoring a protective response were detected in the variables: survival, number ofworms, morbidity and humoral reactivity In conclusion, there were neither evidences of a strong bumoral response nor protection after oral inoculation ofinactivated A. costaricensis larvae.

Abdominal angiostrongyliasis is a zoonotic infection caused by Angiostrongylus costaricensis, a nematode with an intra-vascular location in the mesentery. Our objective was to address several aspects of the natural history of this parasitosis, in a longitudinal clinical and seroepidemiological study. A total of 179 individuals living in a rural area with active transmission in southern Brazil were followed for five years (1995-1999) resulting in yearly prevalence of 28.2%, 4.2%, 10%, 20.2% and 2.8% and incidences of 0%, 5.9%, 8% and 1.5%, respectively. Both men and woman were affected with higher frequencies at age 30-49 years. In 32 individuals serum samples were collected at all time points and IgG antibody reactivity detected by ELISA was variable and usually persisting not longer than one year. Some individual antibody patterns were suggestive of re-infection. There was no association with occurrence of abdominal pain or of other enteroparasites and there was no individual with a confirmed (histopathologic) diagnosis. Mollusks were found with infective third-stage larvae in some houses with an overall prevalence of 16% and a low parasitic burden. In conclusion, abdominal angiostrongyliasis in southern Brazil may be a frequent infection with low morbidity and a gradually decreasing serological reactivity.

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.

Angiostrongylus costaricensisis is an intra-arterial nematode of rodents. Man may become accidentally infected through ingestion of contaminated food or water. Our objective was to describe the parasite structures recognized by human antibodies in serum samples from acute and convalescent phases of abdominal angiostrongyliasis. An indirect immunofluorescent method was employed to study reactivity on whole eggs and sections of female worms and first stage larvae (L1). L1 were also studied before and after sonication. Fluorescence, always higher with acute phase sera, was detected on the surface of whole eggs and in L1 fragments and was neither present on whole L1 nor on their sections. An inespecific reactivity was seen on the cuticular border of the general cavity and reproductive organs. The data indicate the latter as a main source of antigenicity.

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 são 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 sonicação. Fluorescência sempre mais intensa com soros de fase aguda foi detectada na superfície dos ovos inteiros e nos fragmentos de L1 e não estava presente nem nas L1 inteiras, nem em seus cortes. Uma reatividade inespecífica foi detectada na borda cuticular da cavidade geral e sobre os órgãos reprodutores. Os dados indicam que estes órgãos são fonte importante de antigenicidade. Angiostrongylus costaricensisis is an intra-arterial nematode of rodents. Man may become accidentally infected through ingestion of contaminated food or water. Our objective was to describe the parasite structures recognized by human antibodies in serum samples from acute and convalescent phases of abdominal angiostrongyliasis. An indirect immunofluorescent method was employed to study reactivity on whole eggs and sections of female worms and first stage larvae (L1). L1 were also studied before and after sonication. Fluorescence, always higher with acute phase sera, was detected on the surface of whole eggs and in L1 fragments and was neither present on whole L1 nor on their sections. An inespecific reactivity was seen on the cuticular border of the general cavity and reproductive organs. The data indicate the latter as a main source of antigenicity.

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.

Veronicellid slugs are the main intermediate hosts for Angiostrongylus costaricencis. In a rural locality in Nova Itaberaba (SC, southern Brazil) Sarasinula linguaeformis was identified as a crop pest. The parasitological examination revealed A. costaricencis infection in 43 out ot 50 slugs. The prevalence of 86% and the individual parasitic burdens are the highest sofar reported in Brazil and S. linguaeformis is the first species from the genus Sarasinula to be identified as intermediate host for A. costaricencis in southern Brazil. Lesmas veronicelídeas são os principais hospedeiros intermediários de Angiostrongylus costaricencis. Em uma localidade rural de Nova Itaberaba (SC, no sul do Brasil) Sarasinula linguaeformis apresenta-se como peste agrícola. O exame parasitológico das lesmas demonstrou infecção pelo A. costaricencis em 43 de 50 animais. A prevalência de 86% e as cargas parasitárias são as mais altas registradas até o momento no Brasil e S. linguaeformis é a primeira espécie do gênero Sarasinula a ser identificado como hospedeiro intermediário do A. costaricencis no sul do país.

Introduction: Anorexia and hypermetabolism are a problem of paramount importance in children with cirrhosis. Plasma levels of both leptin and ghrelin are altered in patients with starvation and chronic diseases associated with anorexia. Ghrelin has an important role on feeding and weight homeostasis. Aim: We aimed to assess ghrelin and leptin levels in cirrhotic children and adolescents in comparison with controls. Methods: Forty children with cirrhosis (biliary atresia as etiology in 67.5%) aged between 3.7 and 188.7 months, 50% male, were evaluated, 50% were Child-Pugh A; 40%, B and 10%, C. Other 40 healthy children matched for sex and age, taken as controls, were also evaluated. Blood samples were collected at least after 3-hours fasting. Fasting leptin and acylated ghrelin levels were measured using a newly developed ELISA kit (Linco Research, St Charles-MI, USA). Body composition including body mass index (BMI), body fat mass and height for age were determined and classified according to WHO 2009 and Frisancho 2008 standard reference. All controls were eutrophic as well as 77.5% of cirrhotic patients. Other cirrhotics were either undernourished (21.3%) or overweighed (1.3%). Results: Ghrelin and leptin in both groups did not correlate with sex (P=0.023). Median values (25-75 centile) of acylated ghrelin were significantly lower in cirrhotics than in healthy controls [140.3 (93.9-269.7) pg/mL vs 277.8 (209.9-557.8) pg/mL, P=0.003]. Median values of total ghrelin and desacyl ghrelin of children with cirrhosis were 913.4 pg/mL (760.8-1443.7) and 1397.8 pg/mL (815.27-2047) respectively. Median values of leptin were not significantly different between cirrhotics and controls (P=0,26)]. From the cirrhotic group there was a significant difference between undernourished and well-nourished pactients [1.45ng/mL (0.66-1.88) 2.68 ng/mL (1.31-4.38)], P=0.023. Median values of Child-Pugh C were significantly lower than B and A [0.67 (0.17-1.11) ng/mL; 1.83 (1.24-4.04) ng/mL; 1.88 (0.67-3.67) ng/mL], P=0.033. Conclusions: Cirrhotic children showed lower plasma levels of acylated ghrelin compared with controls. This might be representative of the anorexia that consequently contributes to the malnourishment and illness severity of cirrhotic patients.

Objectives This study aimed to establish the relationship between total ghrelin, acyl ghrelin, des-acyl ghrelin, leptin, and insulin with anthropometry, gender, and age distribution in healthy children. Results Data from 111 healthy children aged 4 months to 10 years were studied. All the participants underwent a pre-study screening clinical evaluation and were separated in 3 age groups. All had blood collected to assay. Anthropometric parameters were measured according to World Health Organization. In order to determine the correlation between dependent and independent variables, a multiple linear regression analysis was used. Overall median age of subjects was 60.0 months. After multiple regression analysis, correlation between total ghrelin, acyl ghrelin and des-acyl ghrelin remained significant with age. Correlation between leptin values and age, body mass index-for-age ratio, height-for-age ratio, and female gender remained significant. There was no significant correlation between insulin and ghrelin, and between insulin and leptin in all age groups. There was an inverse significant correlation between total ghrelin and des-acyl ghrelin with leptin in the whole group. Conclusions Ghrelin showed an inverse correlation with age and leptin showed a direct correlation with anthropometric parameters and female gender in healthy children. Insulin did not show any correlation.