Incidência de enteroparasitas com caracterização molecular de Cryptosporidium spp. em diferentes comunidades brasileiras

Abstract

Destas, oito são reconhecidas por diversos pesquisadores como causadoras de criptosporidiose humana: C. parvum (C. parvum genótipo bovino ou genótipo 2 ou genótipo C ou tipo II ou C. pestis)

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

“…Discrepant results between microscopy and PCR were reported by MAGI et al 21 who detected in their samples 20% positivity by PCR, due to the presence of few oocysts distributed in a non-homogeneous form in the analyzed fecal material, similarly to PENG et al 24 with 35.3% positivity suggesting long storage time in formalin of the seventeen fecal samples studied. The general data obtained by PCR (37.23%) in this study resulted extremely positive when compared with those derived from former partnerships performed with protocols sedimented for PCR, in addition to the use of commercial kits for DNA extraction and which resulted in 49.2% and 20.9% amplification of samples 15 . The technique here used demonstrates its utilization in this context, once being associated with RFLP using only two restriction enzymes, is able to distinguish C. hominis and C. parvum (data not shown), responsible for most human infections from other related species, despite failures observed in samples with a low number of oocysts during microscopic screening, which may be overcomed with repetition of extractions.…”

“…Progress in molecular techniques produced a great impact on the knowledge of taxonomy, biology, pathogenesis, epidemiology and transmission of the agent and also on evaluation of cryptosporidiosis treatment, as well as helping epidemiologists regarding control and determination of infection sources 4,5,14,17,30 . Thus, among the molecular methods developed for the detection and differentiation of different Cryptosporidium species and genotypes, PCR is the mostly used for both clinical and environmental samples 8,11,28 , but its sensitivity is affected by the presence of inhibitors existing in fecal samples which may be minimized with changes in the extraction protocol 6,8,10,15,16,22,26 . In Brazil, most laboratories do not identify Cryptosporidium spp.…”

“…In Brazil, most laboratories do not identify Cryptosporidium spp. 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 .…”

“…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…”

“…Cryptosprodium spp. oocysts were detected by the modified Kinyoun method 15 , in smears prepared with 10 µL of fecal sample "in natura" or, in a small proportion and when formed fecal samples, in buffered formalin, on 76 x 26 mm glass slides for microcopy, fixed with methanol PA. When detected, they were measured with the help of an ocular micrometer during reading under a light microscope, 100x objective, and for each slide the mean was calculated.…”