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Doctoral Thesis
DOI
10.11606/T.9.2004.tde-06022015-095557
Document
Author
Full name
Ana Julia Urias dos Santos Araujo
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2004
Supervisor
Committee
Kanamura, Herminia Yohko (President)
Chieffi, Pedro Paulo
Gennari, Solange Maria
Hirata, Rosario Dominguez Crespo
Pinto, Pedro Luiz Silva
Title in Portuguese
Estudo da ocorrência de infecção por Cryptosporidium spp (Apicomplexa: Cryptospordiidae) entre crianças do município de Taubaté- SP e caracterização genotípica de isolados clínicos do parasito
Keywords in Portuguese
Diarreia infantil (Estudo clínico)
Infecções por protozoários (Estudo clínico)
Parasitologia
Abstract in Portuguese
Objetivou-se, com o presente estudo, verificar a ocorrência de Cryptosporidium spp entre crianças do município de Taubaté-SP e realizar a genotipagem de isolados clínicos do parasito. Foram selecionadas 13 creches municipais, que atendiam crianças de 4 a 72 meses de idade, realizando-se implantação de um programa para busca ativa de casos de diarréia, com acompanhamento da população durante o ano de 2002. Para se conhecer o perfil coproparasitológico, realizou-se estudo transversal em quatro das 13 creches selecionadas. Foram examinadas 483 amostras fecais processadas pelo método de concentração em formalina-acetato de etila e, para a visualização de oocistos, esfregaços fecais foram corados pelo método de Kinyoun. No estudo prospectivo, Cryptosporidium spp foi encontrado em 11,1 % (3/27) das amostras, não se evidenciando outras espécies parasitárias. No estudo transversal, foram detectados oocistos em 0,2% (1/456) das amostras e a freqüência para outras espécies parasitárias foi de 30,5% (139/456). No estudo de genotipagem, foram analisadas 14 amostras de humanos, uma de bovino e uma de cão. Dentre os isolados de humanos, quatro eram de crianças de Taubaté-SP, cinco de crianças de uma favela de São Paulo-SP e cinco de pacientes HIV positivos, de Sorocaba-SP. O DNA extraído a partir das amostras fecais foi submetido a Nested-PCR, amplificando-se um segmento de 553pb de um gene que codifica proteína de parede de Cryptosporidium (COWP). Os produtos amplificados foram submetidos a processo de digestão enzimática (RPLP-PCR), e os perfis obtidos por eletroforese evidenciaram três espécies: Cryptosporidium hominis em oito amostras, Cryptosporidium parvum em quatro e Cryptosporidium meleagridis em duas. Dos isolados de Taubaté, dois foram correspondentes a C. hominis e dois a C. parvum. As amostras de bovino e de cão foram positivas para C. parvum. O genótipo dos isolados de Taubaté, humanos e de animais, foram confirmados por análise da sequência do fragmento de 553pb do gene COWP, comparando-se com as sequências disponíveis no GenBank.
Title in English
The occurrence of infection by Cryptosporidium spp (Apicomplexa: Cryptosporidiidae) in children living in city of Taubaté-SP and genotypic characterization of the parasite isolates
Keywords in English
Child diarrhea (clinical study)
Parasitology
Protozoan infections (clinical study)
Abstract in English
The aims of this study were to investigate the occurrence of infection by Cryptosporidium spp in children living in Taubaté, São Paulo, Brazil, and to realize the molecular characterization of the parasite isolates. Thirteen day-care centers were selected and 4-72 months old children were involved in prospective study to detect diarrhea, during the 2002 year. In four of these day-care centers, a transversal study was performed in the beginning of the study to know the prevalence of intestinal parasites of children's community. A total of 483 stool samples were examined by the modified formalin-etil acetate concentration method, and the acid-fast Kinyoun stain was used to visualize oocysts. In the prospective study Cryptosporidium spp were detect in 11.1 % (3/27) of stool samples and no other parasites were found. In the transversal study oocysts were detected in 0.2% (1/456) of the samples and the frequency of infection by other parasites was 30.5% (139/456). In the genotyping study, 14 stool samples from humans and one sample from bovine and another from dog were analyzed. Among the human isolates, four were from children of Taubaté, five were from children living in a slum of São Paulo city and the other five from HIV-positive patients of Sorocaba, São Paulo. The animal samples were collected in Taubaté region. A DNA fragment of 553 pb of the Cryptosporidium oocyst wall protein (COWP) gene was amplified from stool samples by Nested-PCR, and Restriction Fragment Length Polymorphism analysis identified three species: Cryptosporidium hominis in eight samples, Cryptosporidium parvum in four samples and Cryptosporidium meleagridis in two samples. Among samples from Taubaté, two were positive for C. hominis and two for C. parvum. The bovine and dog samples were positive for C. parvum. The findings trom Taubaté were confirmed by sequence analysis of the 553bp amplicons and comparison of the COWP gene available in the GenBank.
 
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2015-02-06
 
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