Toxoplasma gondii has subpopulation structures in different geographical regions caused by less frequent sexual recombination, population sweeps, and biogeography. The majority of strains isolated in North America and Europe fall into one of three clonal lineages, referred to as types I, II, and III. So far, little is known about genetics of Toxoplasma strains in Africa. The present study aimed to determine the genotype of Toxoplasma strains obtained directly from trophoblastic/placental tissues of 29 complicated pregnant women using multilocus nested-PCR-RFLP technique depending on four independent genetic loci (5' SAG2 and 3' SAG2), SAG3, GRA6, and BTUB genes. All samples gave positive amplicons at 5'-3' SAG2 and SAG3 genes. Meanwhile, no amplification products were observed in 12 (41.37%) and 10 (34.48%) samples with GRA6 and BTUB genes, respectively. The restriction pattern revealed the presence of genotype I in all samples, except one sample, which revealed atypical genotype with unusual restriction pattern at 3' SAG2 gene. The negative amplifications in some samples could be due to presence of mutations or polymorphisms in the primer binding sites of these isolates, raising the possibility of mixed or recombinant genotypes. To the best of our knowledge, this is the first time to perform genotype analysis study based on Multiplex nPCR-RFLP technique for genetic characterization of T. gondii in Egypt. Besides, it is the first time to prove that the most prevalent strain of T. gondii, responsible for congenital toxoplasmosis in Upper Egypt, is the highly virulent type I. Atypical genotype was detected as well.

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