Identificação molecular dos isolados de Giardia duodenalis em crianças procedentes dos municípios de Araguari e Uberlândia

Abstract

Giardia duodenalis is a parasite of several mammalian species, including humans, with a worldwide distribution. It is associated with diarrhea and nutritional disorders, especially in children. Molecular characterization of the parasite is crucial to identify assemblages/sub-assemblages related to infection, and its association with clinical manifestations. The goal was to identify the molecular assemblages/sub-assemblages of G. duodenalis in children from pre-school, from Araguari and Uberlândia, Minas Gerais, Brazil. It was used four genes for this research. The cysts were researched with 33% zinc sulfate flotation. The molecular characterization used SSU rRNA, β-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) protocols. We conducted a multilocus genotyping (MLG) and the association between clinical manifestations and assemblage. Cysts of G.duodenalis were found in 45 (19.9%) of 226 stool samples from nurseries children. PCR detection (SSU rRNA protocol) failed to amplify DNA samples. Tpi protocol has amplified thirty-four sequences: 16 assemblage A, 14 assemblage B and four mixed samples A/B. Gdh protocol amplified 32 sequences, including 14 assemblage A, 16 assemblage B and two A/B. For bg protocol 19 samples sequenced, nine was characterized as assemblage A, five as assemblage B, three as E, and two mixed, A/E and B/E. The predominance of assemblages varied with the gene protocol used. Heterogeneous samples were found in all genes, including mixed inter-assemblages A/B, A/E, B/E, and intra-assemblage BIII/BIV. Animal-specific (assemblage E) samples were identified with bg, and not confirmed by other genes. From 45 positive samples by optical microscopy, 35 were amplified by nested PCR for at least one gene. Among all, 12 samples were characterized in full concordance by the three genes. It was described two new MLGs assemblages A and five new MLGs to assemblage B. Assemblage A was highly prevalent in Araguari (p = 0.0101) and assemblage B, in Uberlândia. There was a statistically association (p = 0.0454) between assemblage B and diarrhea. There was no association with other clinical manifestations such as abdominal pain, vomiting, weight loss, flatulence and stool consistency. These findings prove the importance of using more than one gene protocol, since the sensitivity and genetic variability changes with the locus used. Heterogeneous sequences were found for all three genes and were considered mixed infections. PCR failed to amplify positive samples by optical microscopy. The use of MLG was important for the classification of assemblage A isolates, but less effective in assemblage B.