Avaliação da heterogeneidade molecular de Cryptosporidium sp. em amostras clínicas

• Autor(a) principal: Rolando, Roberta Flávia Ribeiro
• Data de Publicação: 2012
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14352
• topic: Cryptosporidium; genotyping; PCR real-time; Automatic sequencing; Molecular epidemiology; Cryptosporidium; genotipagem; PCR em tempo real; Seqüenciamento automático; Epidemiologia molecular; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA::MICROBIOLOGIA MEDICA

Cryptosporidium is a coccidia parasite known to cause diarrhea in humans and animals worldwide. The genus comprises at least 20 confirmed species, with C. hominis and C.parvum major species that cause cryptosporidiosis in humans. Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotypes and subtype levels. The objective of this study was to evaluate the molecular heterogeneity of Cryptosporidium sp. clinical samples obtained from Rio de Janeiro and Salvador (Bahia), through real-time PCR and automatic sequencing. We analyzed 85 samples, distributed in three distinct groups, 45 of them in the city of Rio de Janeiro and 40 samples from Salvador. All samples were positive for Cryptosporidium sp. by modified Kinyoun acid-fast staining technique. The real-time PCR procedure combined a duplex reaction for the detection of Cryptosporidium species and C. parvum and a simple reaction for the detection of C. hominis. The detection of the genus Cryptosporidium have been used a pair of primers and TaqMan probe designed from the alignment of conserved sequences of the 18S rRNA gene of several species of Cryptosporidium available in GenBank. For the detection of species C. parvum and C. hominis were used specific primers and probes derived from sequences of each species available in the GenBank. Detection of Cryptosporidium sp. by 18S rRNA probe, in the duplex reaction, was visualized in 63 of 85 total samples. Of these, the C. parvum TaqMan probe detected 6 samples and the C. hominis TaqMan probe detected 42 samples. Fifteen samples could not be detected by C. hominis or C. parvum probes. In the 18S PCR assays, 31 samples were positive and 27 of them sequenced. Phylogenetic analysis confirmed the presence of C. hominis, C. parvum and C. felis. The analysis of the phylogenetic tree obtained by Neighbor Joining showed that the sequences obtained in this study were grouped with Cryptosporidium species described in the GenBank, with 99% or 100% similarity. Both methods are important tools for the diagnosis of cryptosporidiosis and differentiation of C. hominis and C. parvum in epidemiological investigations, as well as evaluation of Cryptosporidium sp. molecular heterogeneity