Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples

Abstract

Genetic analysis of the SAG2 locus was performed to determine the prevalence of the different genotypes of Toxoplasma gondii (strain types I, II, and III) associated with human toxoplasmosis in Spain. This determination was made directly from primary clinical samples, obviating the previous process of isolation in mice or cell culture. A total of 34 isolates of T. gondii, collected from immunocompromised patients and congenital infection cases, were analyzed. Restriction fragment length polymorphism in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Complete characterization of the SAG2 gene was successful in 76.5% of the cases, demonstrating the feasibility of direct genotype analysis from clinical samples of different origins. Strains of T. gondii type II were the most prevalent in immunocompromised patients, with 52% of cases, while strains of type I were present in 75% of the congenital infection cases. These data differ from previous reports that show type II strains to be mostly associated with all kinds of human toxoplasmosis. These differences might be an effect of selection in the process of culture and isolation of the samples performed by other researchers prior to strain characterization.

FIG. 1

Agarose gel electrophoresis analysis of SAG2 PCR amplification products and restriction digests from T. gondii-infected clinical samples. (A) Sau3AI restriction analysis of the 5′ amplification products and (B) HhaI restriction analysis of the 3′ amplification products. Lane I, DNA from strain RH (type I); lane II, DNA from strain Beverly (type II); lane III, DNA from strain C56 (type III). Lanes 1 to 8 contain strains from clinical samples as follows: lane 1, CSF from patient 1 IHIV+ (type I); lane 2, CSF from patient 5 IHIV+ (type II); lane 3, blood from patient 7 Cnewborn (type II); lane 4, blood from patient 5 Cnewborn (type I); lane 5, CSF from patient 14 IHIV+ (type III); lane 6, vitreous humor from patient 12 IHIV+ (type II); lane 7, blood from patient 8 Cnewborn (type III); lane 8, brain (from biopsy) from patient 8 IHIV+ (type II). Lane M, molecular weight markers.