Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome

Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5’and 3’ SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 10(2) parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 10(2) and ≤ 10(3) (one mild, 13 moderate and three severe), and three had load > 10(3) parasites/mL (three severe); G2 with 22 samples (5’and 3’ SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 10(2) parasites/mL (18 asymptomatic and four mild); G3 with five samples (5’ and 3’ SAG2 allele I + SAG3 allele II), parasite load ≤ 10(2) parasites/mL (three asymptomatic and two mild); G4 with one sample (5’ and 3’ SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 10(2) parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5’-SAG2 allele I; 98.7% of 3’-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.