Leishmania parasites have variability of species in the Amazon region and its correct identification is necessary to isolate them. Currently for the isolation of the parasite and subsequent diagnosis of the disease have used the technique of in vitro microculture. Hence, the aim of our study was to optimize the in vitro microculture technique for isolation of Leishmania sp. to contribute to the identification of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis circulating in the state of Pará For isolation beyond the microculture was analysed the technique of vacuum aspiration adapted, and parasite viability at temperatures under 25°C. Was used 18 hamsters infected with samples from clinical cases of CL, 3 L. (L). amazonensis 2 and L. (V). braziliensis which held 56 culture by vacuumaspiration in NNN media, 12 in microtubes and 23 in microcapillaries with RPMI media supplemented with 10% fetal bovine serum and 1% Penicillin-Gentamicin maintained between 25°C and 31°C. For the second stage, participated in seven patients, a total of 6 cultures by vacuum aspiration and 42 by microcapillary. It was remained at low temperature 7 tubes with NNN which were kept at 5°C. It was observed that the isolates by vacuumaspiration samples of L. (L.) amazonensis and L. (V.) braziliensis in hamsters were susceptible to adaptation of the technique, differently of samples of patients. The positivity ranged between 2-8 days and 4 and 5 days respectively. The microtubes were positive for the same samples of hamsters in the period 5-8 days. For samples of patients, 2/12 tubes by vacuum-aspiration were positive for isolation and in microcapillaries 6/42 less than the values found in literature. The samples stored at 5°C showed viability until 30º day. Thus, we find that the microculture is viable for use within our region.
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