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Allelic variants of t. gondii ROP18 and ROP5 roptries in cases of congenital toxoplasmosis: association with pathogenicity in mice and clinical outcome

Grant number: 18/06482-0
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): September 01, 2018
Effective date (End): August 31, 2021
Field of knowledge:Health Sciences - Medicine
Principal Investigator:Thelma Suely Okay
Grantee:Larissa Gabrielle Lima de Araújo Curval
Home Institution: Instituto de Medicina Tropical de São Paulo (IMT). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Toxoplasmosis affects at least one third of the world's population and is usually benign, except for infection in patients with some degree of deficiency of the immune system and in fetuses and neonates due to their immunological immaturity. The severity of congenital toxoplasmosis is influenced by several factors such as the gestational age at which the fetus was infected, the genetic susceptibility of the host, the parasite load on amniotic fluid and possibly the parasite genotype. Among the genotyping techniques, the most used is the multilocus-nested-PCR of 11 markers, but not all markers amplify samples that were positive in diagnostic tests. In addition, these markers generate an excessive number of genotypes in South America, without association with infections severity. In an attempt to improve the efficiency and reproducibility of T. gondii genotyping, we propose the development of three nested-PCR sequences from roptries 18 and 5 followed by Sanger sequencing, instead of two RFLPs performed with four restriction enzymes (double digestions). Three hundred and fifteen biological samples (amniotic fluid, fetal blood, placenta, neonatal blood, cerebrospinal fluid, ocular fluid) from confirmed cases of congenital toxoplasmosis, belonging to a biorepository, will be tested. Regarding the alleles found in ROP18 and ROP5, we will verify the pathogenicity thas has already been described for them in mice and the outcome of the infections of pregnant women and infants (determined by monitoring of the pregnant women until delivery and of the children for 12 months). It is also intended to compare the efficiency and resolution of the ROP18 and ROP5 amplifications in relation to some of the markers used in multilocus-nested-PCR-RFLP, notably 5 'and 3' SAG2, SAG3 and GRA6.