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

Abstract

The aims of this study were to investigate the occurrence of infection b Cryptosporidium spp in children living in Taubaté, São Paulo, Brazil, and to realiz the molecular characterization of the parasite isolates. Thirteen day-care cente were selected and 4-72 months old children were involved in prospective study detect diarrhea, during the 2002 year. In four of these day-care centers, transversal study was performed in the beginning of the study to know th prevalence of intestinal parasites of children's community. A total of 483 sto samples were examined by the modified formalin-etil acetate concentration metho and the acid-fast Kinyoun stain was used to visualize oocysts. In the prospectiv study Cryptosporidium spp were detect in 11.1 % (3/27) of stool samples and n 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 samp from bovine and another from dog were analyzed. Among the human isolates, fo were from children of Taubaté, five were from children living in a slum of São Pau city and the other five from HIV-positive patients of Sorocaba, São Paulo. Th animal samples were collected in Taubaté region. A DNA fragment of 553 pb of th Cryptosporidium oocyst wall protein (COWP) gene was amplified from sto samples by Nested-PCR, and Restriction Fragment Length Polymorphism analys identified three species: Cryptosporidium hominis in eight samples, Cryptosporidiu parvum in four samples and Cryptosporidium meleagridis in two samples. Amon samples from Taubaté, two were positive for C. hominis and two for C. parvum. Th bovine and dog samples were positive for C. parvum. The findings from Tauba were confirmed by sequence analysis of the 553bp amplicons and comparison the COWP gene available in the GenBank.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

“…oocysts routinerely 7,15 and there is no standardization of molecular techniques for genotypic characterization in isolated samples of fecal material. This suggests that information about circulating species and genotypes are scarce in our country 1,2,3,6,12,13,14,15 . Preservation of fecal samples for molecular analysis may be performed in 2.5% potassium dichromate at 4 °C 10,12,14,18 , in absolute ethanol at room temperature 19,22 , in 10% formalin 18,31 , under refrigeration at 4 °C…”

"Padronização e avaliação de métodos moleculares para detecção de oocistos de Cryptosporidium spp. (Apicomplexa: Cryptosporiidae) em amostras fecais: extração de DNA genômico e PCR (reação em cadeia pela polimerase)"

“…No Brasil há três pesquisas acadêmicas que fizeram uso da metodologia descrita por da , empregando o FastDNA ® kit (BIO 101, Inc., Vista, CA) para extração do DNA e o QIAquick PCR purification kit (Qiagen Inc., Santa Clarita, CA) para remoção dos inibidores de PCR (GONÇALVES 2002;GOMES 2003;ARAUJO 2004).…”

Avaliação de métodos de extração de DNA de Cryptosporidium spp. em amostras fecais e comparação de nested PCR com o médodo coproparasitológico de centrífugo-flutuação em sacarose

“…A maior parte dos casos de criptosporidiose em uma cidade urbana nos EUA foi devido à espécie C. hominis (>75%) (SULAIMAN et al, 1998), enquanto que C. parvum foi responsável por 61,5% dos casos em uma cidade rural do Reino Unido (MCLAUCHLIN et al, 2000). No Brasil, também foram relatadas as ocorrências relacionadas a C. parvum em pacientes residentes em áreas peri-urbanas e C. hominis, em pacientes de áreas estritamente urbanas (BRANTLEY, 2003;ARAÚJO, 2004).…”