Molecular typing of Toxoplasma gondii. Analysis of amniotic fluid samples from pregnancies with diagnosis of congenital toxoplasmosis / Tipagem molecular de Toxoplasma gondii: análise de líquidos amnióticos de gestações com diagnóstico de toxoplasmose congênita

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Toxoplasma gondii is the etiologic agent of toxoplasmosis and may cause different forms of the disease, including congenital toxoplasmosis whose frequency varies significantly according to the country. In France, it was estimated in 1-3 cases/1,000 live births, while in the U.S. it is 1/10.000. The prevalence of congenital infection in Brazil is unknown. Toxoplasma gondii has three clonal lineages known as genotypes I, II and III which are related to the three types described according to the pathogenicity in mice, associated with human infections of high pathogenicity, low pathogenicity and intermediate pathogenicity, respectively. The three genotypes have been identified in North America and Europe, and the genotype II is the predominant one in congenital infections. This study aimed at determining the frequency of genotypes I, II and III of T. gondii in 85 amniotic fluid samples from pregnant women living in Sao Paulo, Brazil who presented with seroconversion to Toxoplasma gondii during gestation. The multiplex-nested- PCR-RFLP was performed using four different markers: 3-SAG2, 5-SAG2, SAG3 and GRA6. Eighty-five samples were initially included, but only 76 were positive in the multiplex-nested-PCR (92.7%). It was possible to examine the larger number of samples by the SAG3 gene system (69.7%), followed by 3-SAG2 gene (44.7%), 5-SAG2 gene (40.8%), and finally GRA6 gene (25%). From the 76 samples that were genotyped by the multiplexnested- PCR-RFLP, 1.3% (1 sample) was genotype I; 71.1% (54 samples) were genotype II; 6,6% (5 samples) were genotype III, and 21% (16 samples) could not be identified by RFLP and were therefore sequenced. Sequencing analysis revealed: two genotype II samples, presenting with 100% of homology with the ME49 prototype, and two were genotype III showing 100% of homology with the VEG prototype. Ten samples presented with mixture of two or more nucleotide bases in one or more nucleotide positions of the total amplified DNA sequence, so they were classified as recombinant. In addition, six samples identified by the multiplex-nested-PCR-RFLP as type II were randomly selected to confirm the results, and in four of the six samples, we observed a mixture of nucleotide bases in positions that were not analyzed by the RFLP. The technique of multiplex-nested-PCR-RFLP has proved to be useful, however, it is limited when amniotic fluid samples genotyping is concerned because after sequencing, several genetic recombinations were evidenced in samples that had been previously identified as genotype II. Therefore, we suggest that the genotyping of T. gondii, directly from biological samples or after isolation of the parasite should be performed using different targets to amplify multiple T. gondii genes followed by sequencing of all amplified fragments

ASSUNTO(S)

toxoplasmosis congenital toxoplasmose congênita tipagem de dna reação em cadeia da polimerase infection infecção toxoplasma gondii toxoplasma gondii genotyping polymerase chain reaction

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