Serological and molecular investigation of Toxoplasma gondii in blood donors

Abstract

Infection with Toxoplasma gondii, the etiological agent of toxoplasmosis, worldwide distribution and affects humans of all ages and ethnicities. The spread of the infection by different ways, including transfusion. In immunocompetent individuals, this parasite does not offer great risks, but in individuals imuncomprometidos involvement can be severe and result in high mortality. Objectives. The goal of this project is to detect the use of serological methods and describe the use of molecular methods to infection by T. gondii in blood donors. Specific objectives include: 1. Perform serological screening for anti-T. gondii (IgM and IgG) in suitable candidates for blood donation and identify their profiles [(IgM non-reactive, non-reactive IgG); (IgM, IgG non-reactive); (IgM, IgG reagent); (IgM nonreactive; IgG reagent)]. 2. Detecting the presence of genomic DNA of T. gondii in the peripheral blood of donors with the use of gene amplification methods (conventional PCR and real-time PCR). 3. Characterize the donors with positive PCR, the genotypes of T. gondii with the use of molecular markers. 4. Track units of blood that are experiencing positive for IgM and CRP (cn PCR and RT-PCR) and their receptors with the use of these serological and molecular methods. Patients and methods. Will be selected at least 2000 blood donors are able to donate at the Blood Center of São José do Rio Preto, São Paulo State. For each donor will be filled with a plug and epidemiological data are collected two samples of peripheral blood at the time of donation. One will be used for the detection of antibodies (IgM and IgG) anti-T. gondii, using the ELISA method. The other will be used for genomic DNA extraction using commercial kits. The gene amplification of T. gondii will be performed by conventional PCR (cnPCR) and real-time PCR (qPCR). The positive samples are genotyped by PCR using the markers SAG1, SAG2 (50-and 30-SAG2, alt.SAG2) SAG3, BTUB, GRA6, C22-8, c29-2, L358, and apical PK1. As the serological profile, obtained from ELISA, blood donors will be separated into four groups (G1, G2, G3 and G4). G1 will be included in the donor profile serological IgM and IgG non-reactive. In G2, are included those with IgM and IgG reagent; G3 in those IgM and IgG non-reactive, and G4 nonreactive IgM and IgG reagent. All these groups will be compared with the result of PCR for T. gondii. The results obtained and the PCR will be compared by using the Ç2 test and Fisher's exact test, when appropriate. Also be calculated Odds Ratio values and confidence intervals at 95%. Expected results. It is hoped that the results to be achieved with this project will contribute to meeting the T. gondii circulating in the peripheral blood of donors and to estimate the risk that the presence of such parasites provides patients requiring transfusion. (AU)