Complementary diagnosis

When the standard serological tests had inconclusive results, other confirmatory exams were requested by the specialized service, including the anti- T. gondii IgA serological test, performed by the enzyme-linked immunosorbent assay (ELISA) method (reference values: <0.80, negative; > 1.10, positive) from an outsourced service. Tests for detection of T. gondii DNA in peripheral blood and amniotic fluid samples, as well as inoculation in animals, were performed, in some cases, by the Parasitology Laboratory, State University of Maringa.

Polymerase chain reaction (PCR) amplifications were performed with DNA extracted from amniotic fluid and peripheral blood samples by using the commercial kit Purelink^® (Thermo Fisher Scientific, Waltham, USA), with primers from the B1 gene and Toxo 4-5); and samples were also intraperitoneally inoculated in mice 22 .

T. gondii DNA genotyping was carried out on amniotic fluid and blood samples from pregnant women or mice specimens using the genetic markers SAG1, 5’-3’SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Strains GT1, PTG, CTG, TgCgCa1, MAS, TgCatBr5 and TgCatBr64 were used as standards 23 . The target DNA sequences were amplified by multiplex-PCR with external primers for all the markers, followed by nested-PCR using internal primers for each marker individually. The results were compared and ranked according to the ToxoDB database ( http://toxodb.org/toxo/ ).

The animal experiments were approved by the Ethical Committee for Animal Use of the State University of Maringa (CEUA/UEM N° 036/2009 and 5654290317, 2017).

All pregnant women (including confirmed or suspected cases) underwent imaging diagnosis and they were monitored monthly until the end of gestation by ultrasonography (EnVisor HD with high-definition convex probe, 2-5 MHz; Philips EnVisor, Riverside, CA, USA).