Programa de Pós-graduação em Biologia da Relação Parasito-Hospedeiro
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Navegando Programa de Pós-graduação em Biologia da Relação Parasito-Hospedeiro por Por Orientador "Dorta, Miriam Leandro"
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Item Leishmaniose Tegumentar Americana na região Centro-Oeste: avaliação de dados clínicos, epidemiológicos, laboratoriais e moleculares(Universidade Federal de Goiás, 2014-04-30) Balian, Rosana Pereira Morais; Dorta, Miriam Leandro; http://lattes.cnpq.br/3933395097851681; Dorta, Miriam Leandro; Moraes, Sandra do Lago; Loyola, Patrícia Resende Alo NagibProtozoa of the genus Leishmania, which affects the skin and/or mucous membranes, cause American Cutaneous Leishmaniasis (ACL). It is an endemic zoonosis whose numbers of cases in the Brazil Midwest region are growing. In the Goiás state, 2798 ACL cases were been reported between 2007 and 2013. In the present study, our proposal was to investigate the epidemiological and molecular characteristics of ACL patients attended at the ambulatory of the Hospital Anuar Auad in the period 2000 at 2006 and identify the species of Leishmania sp. The study included 152 patients with ACL, 124 were from of the state of Goiás and 28 at Mato Grosso, aged between 6-79 years, and center and thirteen these individuals were male. For diagnosis, clinical, epidemiological and laboratory data were collected, such as direct examination (ED), histopathology (AH), Montenegro skin test (MST), indirect immunofluorescence (IIF). ELISA using crude extract of L. (Viannia) braziliensis was performing. Characterization of Leishmania species was carried out by polymerase chain reaction (PCR). The positivity of ED, AH, MST, IIF and ELISA was 70.6%, 80.9%, 68.9%, 44.2% and 73.0%, respectively. Specific IgG to L. (V.) braziliensis were detecting in 84.7% of patients with mucosal leishmaniasis (ML), significantly higher than those found in patients with cutaneous leishmaniasis (CL), which was 69.0% (p < 0.05). Detection of IgG before and after treatment was performed using ELISA and was observed a statistically significant difference only in samples obtained from patients with CL after 6 and 18 months of treatment. The data serological monitoring of patients with ACL before and after treatment indicates that the total IgG levels tend to decrease after 6 months of treatment. PCR was performed on 69 samples obtained from scraping the edge of lesions of patients with ATL, these, 62 (89.8%) were positive, 53 patients with LC and 9 patients with ML. The samples were characterized by PCR as L. (V.) braziliensis (93.5%) and L. (L.) amazonensis (6.5%). It was found that 25.8% of cases of leishmaniasis in the state of Goiás occurred in Campestre, Goiânia, Aparecida de Goiânia and Niquelândia. By analyzing the frequency of cases of ATL over the months during the years analyzed, it was observed that there was a distribution of ACL in all seasons from 2000 to 2006, with a larger number of cases in winter. PCR represents important and powerful tool in the diagnosis and species identification in ACL in endemic areas. ACL controlling in endemic areas is difficult and requires an accurate idea of its epidemiology.Item Padronização da análise dos produtos da PCR/RFLP que amplifica os genes Ribossomal Internal transcribed spacer (ITS) e Glucose-6- phosphate dehydrogenase (G6PD) para identificação de Leishmania spp. em gel de poliacrilamida(Universidade Federal de Goiás, 2017-08-30) Marques, Cálita Pollyanna; Dorta, Miriam Leandro; http://lattes.cnpq.br/3933395097851681; Dorta, Miriam Leandro; Soares, Joanna Darc A. Herzog; Gomes, Rodrigo SaarsAmerican Cutaneous Leishmaniasis (LTA) is caused by protozoa of the genus Leishmania and it attacks the skin and/or mucous membranes. It is an endemic zoonosis in Brazil being notified in all the Regions. There is a relation between the species of parasite and the clinical manifestations, contributing to the diagnostic complexity, which up to the present, did not exist species-specific diagnosis. In the present work, the proposal was to standardize the analysis of the PCR/RFLP products to the ITS and G6PD genes in polyacrylamide gel. In order to standardize PCR/RFLP analysis on the ITS and G6PD genes, DNA from the reference Leishmania spp. Sample was used and 5%, 6%, 7% 8% and 10% polyacrylamide were tested. The protocol design considered the size of the amplicons generated by the PCR / RFLP, correlating the concentration of the polyacrylamide. To standardize the electrophoresis time the migration of the dyes used in the DNA sample buffer was monitored. To validate the technique, we selected 521 samples from Tocantins patients fixed on slides and 43 samples of parasite isolates stored in Leishbank. The samples from the patients from Tocantins were analyzed by PCR/RFLP to the kDNA and were selected to those positive for the validation. The concentration of the matrix for analysis of the PCR and RFLP products to the ITS gene was standardized in 6% and 8%, respectively, and distinguished the species L. (L.) amazonensis, but the species L. (V.) braziliensis, L. (V.) guyanensis and L. (V.) lainsoni were not distinguished among themselves, occurring the same between L. (V.) naiffi and L. (V.) shawi. The concentration of the matrix for analysis of the PCR products to the G6PD gene was standardized in 6% and distinguished L. (V.) braziliensis from the others. The validation of the polyacrylamide gel analysis of the PCR products to the ITS target from samples of patients fixed in slides was not performed, since of the 256 samples analyzed only 4 amplified. The validation of the analysis of the products of the PCR to the G6PD target in polyacrylamide gel from 48 samples fixed in slides characterized 81% as L. (V.) braziliensis and, characterized 63% of the isolates of parasites as L. (V.) braziliensis. The PCR/RFLP analyzes performed to discriminate Leishmania spp. are complementary and thus contribute to the species-specific diagnosis of LTA.