Molecular Characterization Of Intestinal Protozoa In Two Poor Communities In The State Of São Paulo, Brazil.

Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.810

Molecular characterization of intestinal protozoa in two poor communities in the state of Sao Paulo, Brazil

Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tiete in the State of Sao Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis8COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2011/52100-

Evaluation of the presence of Cryptosporidium spp. and Giardia spp. in natural mineral waters from springs and enteroparasitosis in two rural communities in the city of Campos do Jordão, S.P., Brazil

Orientador: Regina Maura Bueno FrancoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A presença de espécies dos protozoários Cryptosporidium e de Giardia em água, principalmente as destinadas ao consumo humano, assumiu nos últimos 22 anos grande importância em Saúde Pública devido à veiculação hídrica de suas formas infectantes. Esses protozoários causam gastroenterites no ser humano e em diversas espécies animais. A transmissão desses protozoários ocorre pela via fecal-oral mediante a ingestão de água ou de alimentos contaminados, ou pelo contato direto (pessoa-a-pessoa ou animal-pessoa; transmissão zoonótica). A persistência de oocistos e cistos no ambiente e a grande resistência aos processos de desinfecção contribuem com a veiculação hídrica desses protozoários. Numerosos surtos de gastroenterite aconteceram nos últimos 25 anos devido à ingestão de água de consumo e involuntariamente águas recreacionais contaminadas. No Brasil é crescente a degradação de águas superficiais e subterrâneas devido à deficiência de infra-estrutura no sistema de esgotamento sanitário. Análises realizadas pela Sabesp sobre a qualidade microbiológica da água proveniente das fontes naturais da cidade de Campos do Jordão (nordeste do estado de São Paulo) revelam que 13, de um total de 22 fontes, apresentaram padrão de qualidade impróprio para o consumo humano ¿Observação: O resumo, na íntegra poderá ser visualizado no texto completo da tese digitalAbstract: The presence of protozoan parasites Cryptosporidium and Giardia mainly in water destined to the human consumption, assumed in the last 22 years, a great importance in Public Health due to the water transmission of its infectant forms. These protozoa cause gastroenteritis in human and in many animal species. The transmission of these parasites occurs by oral-fecal route, consumption of contaminated water or food or by the direct contact (person to person, animal to person or zoonotic transmission). The persistence of oocysts and cysts in the environment and its resistance to the conventional process of disinfection contribute to the waterbome transmission of these protozoa. Numerous outbreaks of gastroenteritis occurred in the last 25 years due to the consumption water and involuntarily recreational waters contaminated. In Brazil, the degradation of subterranean and superficial waters is growing due to the deficiency of infrastructure in the sanitary system. Analyses carried out by the Sabesp about the microbiological quality of the water originating from the natural springs of the city of Campos do Jordão (northeast of the state of São Paulo) revealed that 13, out of a total of 22 springs, presented standard of quality unfit for the human consumption ...Note: The complete abstract is available with the full electronic digital thesis or dissertationsMestradoParasitologiaMestre em Parasitologi

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in the water sources used for the urban supply of Campinas, São Paulo, Brazil and comparision of two filtration methods for the detection of these protozoa

Orientadores: Regina Maura Bueno Franco, Romeu Cantusio NetoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Dados sobre a ocorrência das diferentes espécies de Cryptosporidium e das assembleias de Giardia duodenalis em amostras de água bruta no Brasil são escassos. A escolha de um método capaz de detectar a presença desses protozoários nos mananciais é de extrema importância epidemiológica. O objetivo desse estudo foi realizar a caracterização molecular de cistos e oocistos detectados nos rios Atibaia e Capivari que são os principais mananciais que abastecem o município de Campinas, SP. Esses rios mostram-se eutrofizados dado que estão localizados em uma das regiões mais industrializadas do estado de São Paulo. Amostras de água bruta foram filtradas por dois diferentes métodos, sistema Filta-Max® (Iddex) e por Filtração em Membranas de ésteres mistos de celulose (47 mm de diâmetro) na qual adicionou-se uma etapa de eluição aquecida, objetivando-se a comparação entre esses dois procedimentos analíticos quanto à eficiência de recuperação de cistos e oocistos, empregando-se para tanto a suspensão comercial ColorSeed®. Os protozoários foram purificados por separação imunomagnética (IMS) e visualizados com o emprego de anticorpos monoclonais de acordo com o protocolo do Método 1623.1 da Agencia de Proteção Ambiental dos Estados Unidos. Amostras de água bruta de ambos os mananciais (n= 24) foram processadas mediante o método de referencia visando a caracterização molecular dos protozoários potencialmente patogênicos Cryptosporidium e Giardia. Empregou-se os marcadores moleculares SSU rRNA, beta-giardina, glutamato-desidrogenase e o espaçador transcrito interno do gene 5.8S para a caracterização molecular de G. duodenalis e, para Cryptosporidium sp, objetivou-se a amplificação de um fragmento de 830 pares de bases do gene 18S rRNA. Testes estatísticos demonstraram desempenho similar de ambas as metodologias em estudo quanto à eficiência de recuperação de cistos e oocistos; entretanto, esses procedimentos analíticos acarretaram em maior eficiência de recuperação de cistos que oocistos e, a turbidez exerceu efeito prejudicial na performance de ambas as metodologias em estudo como evidenciado pelo achado de correlação negativa entre turbidez e recuperações de cistos e oocistos. Cistos de Giardia sp ocorreram com maior frequência que oocistos de Cryptosporidium sp em ambos os mananciais. Para o Rio Atibaia, houve concordância entre os resultados para o marcador molecular SSU rRNA e visualização por imunofluorescência, sendo 100% das amostras positivas para cistos enquanto que 66,6% das amostras de água do Rio Capivari resultaram em amplificação. No Rio Atibaia, registrou-se a presença de duas espécies de Cryptosporidium quando empregado o marcador molecular 18S rRNA, a saber: C. hominis e C. parvum; a espécie C. meleagridis foi detectada em uma amostra de água bruta do rio Capivari, assim, essas três principais espécies infecciosas para o ser humano foram detectadas nos locais de captação dos mananciais utilizados para abastecimento público no município de Campinas, sendo tal achado relevante do ponto de vista de Saúde Publica. Para Giardia duodenalis, as análises de sequenciamento apontaram para a presença das assembleias AI e BIV nas amostras do rio Capivari e, no rio Atibaia, a ocorrência da assembleia A tendo-se observado diferenças quanto ao sucesso de amplificação, considerando os quatro marcadores moleculares utilizados. Clonagem do DNA de Cryptosporidium permitiu comprovar a presença de C. meleagridis em uma amostra de água do rio CapivariAbstract: Data about the occurrence of various Cryptosporidium species and different Giardia duodenalis assemblages in water samples from Brazil yet are scarce. The selection of a filtration method able to detecting the presence of these protozoan parasites in water sources is of high epidemiological relevance. We investigate the occurrence of these pathogenic protozoa in raw water samples of Atibaia and Capivari River which are the main water supplies in Campinas city, São Paulo State. These rivers show high degrees of eutrophication as they are located in one of the most industrialized regions in São Paulo State. Samples were concentrated by membrane (47 mm of diameter) filtration technique added a heated elution step and/or by USEPA Method 1623.1 (method of reference) using the system Filta-Max®, aiming the comparison between these two analytical procedures through the recovery efficiencies of cysts and oocysts by using the ColorSeed® commercial suspension. Protozoa were purified by Immunomagnetic separation (IMS) and they were visualized by monoclonal antibodies label according with Method 1623.1. Raw water samples from both water sources were processed through the reference method objecting the molecular characterization of pathogenic protozoan parasites Cryptosporidium spp. and Giardia spp. The molecular markers SSUr RNA, beta-giardin, glutamate-desidrogenase and internal transcribed spacer of 5.8 gene were employed for the molecular characterization of G. duodenalis; for Cryptosporidium, the amplification of an 830 bp fragment of the 18S rRNA gene was used. Statistical analysis showed similar performance of both methodologies under study for cysts and oocysts recovery efficiencies; however, these analytical procedures resulted in a higher recovery efficiency of cysts than oocysts, and turbidity had a detrimental effect on performance of both methodologies as evidenced by our findings (negative correlation between turbidity and cysts/oocysts recoveries). Giardia sp cysts occurred more frequently than Cryptosporidium sp oocysts in both water sources. For Atibaia River, there was agreement between the results for SSU rRNA molecular marker amplification and immunofluorescence assay visualization, with 100% of samples positive for cysts while 66.6% of water samples from Capivari River resulted in amplification. In Atibaia River, the presence of two Cryptosporidium species was documented when the molecular marker 18S rRNA was utilized, namely: C. hominis and C. parvum; C. meleagridis was detected in a sample of raw water of Capivari River; thus, these three main infectious species for humans were detected at the catchment sites of water sources intended for public supply in Campinas city. These findings are relevant from a public health point of view. For G. duodenalis, sequencing analysis pointed to the presence of AI and BIV assemblages in raw water from Capivari River, and in the Atibaia River, the occurrence of assemblage A was detected. In this study, differences were observed regarding the success of amplification considered the four molecular markers used for G. duodenalis. Cloning of DNA of Cryptosporidium allowed to confirm the presence of C. meleagridis in one Capivari River water sampleDoutoradoRelações Antrópicas, Meio Ambiente e ParasitologiaDoutor em Biologia Animal2012/50522-0FAPES

Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS

A toxoplasmose cerebral pode ser altamente debilitante e ocasionalmente fatal em pessoas com deficiências do sistema imune. Nesse estudo, nós avaliamos a resposta de anticorpos das subclasses da IgG para o Toxoplasma gondii em 19 amostras de líquido cefalorraquidiano (LCR) de pacientes com toxoplasmose cerebral que apresentavam uma reação IgG anti-T. gondii positiva com ELISA padronizada com uma preparação antigênica de cistos. Não foram encontradas diferenças significativas entre as taxas de positividade e as concentrações de anticorpos (média aritmética das absorbâncias das reações ELISA, MEA) para IgG1 e IgG2, mas as taxas de positividade e valores MEA para estas duas subclasses de IgG foram significativamente superiores aos da IgG3 e IgG4. A resposta marcante de anticorpos IgG2 em LCR de pacientes com toxoplasmose cerebral merece investigação adicional.Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation

Morphological assessment of dentine and cementum following apicectomy with Zekrya burs and Er:YAG laser associated with direct and indirect Nd:YAG laser irradiation

Objectives. This study aimed to assess the apical surface morphology of maxillary central incisors resected 3.0 mm from the tooth apex using Zekrya burs or Er:YAG laser, with or without subsequent direct Nd:YAG laser irradiation (apical and buccal surfaces) and indirect irradiation (palatal surface). Study design. Forty maxillary central incisors were instrumented and obturated. The roots were divided into 4 groups according to the root resection method (Zekrya bur or Er: YAG laser -1.8 W, 450 mJ, 4 Hz, 113 J/cm(2)) and further surface treatment (none or Nd: YAG laser -2.0 W, 100 mJ, 20 Hz, 124 J/cm(2)). The teeth were prepared for SEM analysis. Scores ranging from 1 to 4 were attributed to cut quality and morphological changes. The data were analyzed by the Kruskal-Wallis test and by Dunn`s test. Results. SEM images showed irregular surfaces on the apical portions resected with Zekrya burs, with smear layer and grooves in the resected dentine and slight gutta-percha displacement and plasticization. On the other hand, apicectomies carried out with Er: YAG laser showed morphological changes compatible with ablated dentine, with rough surfaces and craters. In spite of the presence of plasticized gutta-percha, with the presence of bubbles, an irregular adaptation of the filling material to the root walls was also observed. Direct Nd: YAG laser irradiation of the apical and buccal surfaces of the resected roots resulted in areas of resolidification and fusion in the dentine and cementum, with a vitrified aspect; indirect Nd: YAG laser irradiation of the palatal surfaces yielded a lower number of changes in the cementum, with irregular resolidification areas. Conclusions. There were no differences in terms of cut quality between the use of burs and Er: YAG laser or between the 2 surfaces (apical and buccal) treated with Nd: YAG laser with direct irradiation. However, morphological changes were significantly less frequent on surfaces submitted to indirect irradiation (palatal) when compared with those directly irradiated. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: e77-e82

Layer-by-layer assembly of functionalized reduced graphene oxide for direct electrochemistry and glucose detection

We report an electrochemical glucose biosensor made with layer-by-layer (LbL) films of functionalized reduced graphene oxide (rGO) and glucose oxidase (GOx). The LbL assembly using positively and negatively charged rGO multilayers represents a simple approach to develop enzymatic biosensors. The electron transport properties of graphene were combined with the specificity provided by the enzyme. rGO was obtained and functionalized using chemical methods, being positively charged with poly(diallyldimethylammonium chloride) to form GPDDA, and negatively charged with poly(styrene sulfonate) to form GPSS. Stable aqueous dispersions of GPDDA and GPSS are easily obtained, enabling the growth of LbL films on various solid supports. The use of graphene in the immobilization of GOx promoted Direct Electron Transfer, which was evaluated by Cyclic Volt-ammetry. Amperometric measurements indicated a detection limit of 13.4 mu mol.L-1 and sensitivity of 2.47 mu A.cm(-2). mmol(-1).L for glucose with the (GPDDA/GPSS)(1)/(GPDDA/GOx)(2) architecture, whose thickness was 19.80 +/- 0.28 nm, as determined by Surface Plasmon Resonance (SPR). The sensor may be useful for clinical analysis since glucose could be detected even in the presence of typical interfering agents and in real samples of a lactose-free milk and an electrolyte solution to prevent dehydration. (C) 2016 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Sao Paulo State Univ UNESP, Sao Paulo, BrazilUniv Fed Sao Carlos, UFSCar, Campus Sorocaba, Sao Carlos, SP, BrazilUniv Fed Sao Paulo, Unifesp, Campus Sao Jose dos Campos, Sao Paulo, SP, BrazilUniv Campinas Unicamp, Sao Paulo, BrazilInstituto de Ciência e Tecnologia, Universidade Federal de São Paulo UNIFESP, Rua Talim, 330, 12231-280 São José dos Campos, SP, BrazilFAPESP: 2014/17519-1FAPESP: 2014/15093-7Web of Scienc