Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological testing of samples from infants in Poland diagnosed with congenital toxoplasmosis

Abstract

Toxoplasma gondii has a clonal population genetic structure with three (I, II, and III) lineages that predominate in North America and Europe. Type II strains cause most cases of symptomatic human infections in France and the United States, although few other regions have been adequately sampled. Here we determined the parasite genotype in amniotic fluid and cerebrospinal fluid samples from congenital toxoplasmosis cases in Poland. Nineteen confirmed congenital cases of toxoplasmosis were analyzed, including both severe and asymptomatic cases. The genotype of parasite strains causing congenital infection was determined by direct PCR amplification and restriction fragment length polymorphism analysis. Nested multiplex PCR analysis was used to type four independent polymorphic markers. The sensitivity of multiplex nested PCR was >/=25 parasites/ml in amniotic fluid and cerebral spinal fluid samples. Parasite DNA was successfully amplified in 9 of 19 samples (eight severely affected and one asymptomatic fetus). Only genotype II parasites were identified as the source of T. gondii infection based on restriction fragment length polymorphism analysis. Strains causing congenital infections were also typed indirectly based on detection of antibodies to strain-specific peptides. Serotyping indicated that 12 of 15 cases tested were caused by type II strains and these positives included both symptomatic and asymptomatic infections. Overall, the combined analysis indicated that 14 of the cases were caused by type II strains. Our results are consistent with the hypothesis that parasite burden is associated with severity of congenital toxoplasmosis and indicate that serological testing provides a promising method for genotypic analysis of toxoplasmosis.

FIG. 1.

Sensitivity of multiplex PCR analysis of T. gondii in AF (A) and CSF (B) samples. Multiplex PCR was performed for the four genetic markers SAG2 (5′ and 3′ amplified separately), SAG3, GRA6, and BTUB followed by electrophoresis in agarose gels containing ethidium bromide. Lanes 1, 6, and 7 are negative controls, and lanes 2 to 5 are samples spiked with 0.5, 2.5, 5.0, and 10.0 T. gondii cells, respectively.

FIG. 2.

RFLP analysis of PCR products amplified from AF and CSF samples from cases of congenital toxoplasmosis. 3′-SAG2 amplification products digested with HhaI were resolved in 3% agarose gels stained with ethidium bromide. The samples loaded in lanes I, II, and III are representative of T. gondii strain types I, II, and III, respectively. Samples from pregnancies with congenitally infected fetuses were loaded in lanes 1 to 9. Genotyping revealed all nine positive samples were type II strains of T. gondii. AF samples from uninfected pregnant women (neg) served as negative controls.