Serological and molecular investigation of Toxoplasma gondii in blood donors

Abstract

Introduction. Infection with Toxoplasma gondii, the etiological agent of toxoplasmosis, has a worldwide distribution and affects humans of all races and age. The spread of infection can occur in different forms, including the transfusion of blood products. In immunocompetent individuals this parasite does not offer big risks, but in immunocompromised individuals involvement can be severe and may have a high mortality rate. Objectives. The objective of this project is to detect the use of serological methods and characterization using molecular methods to infection by T. gondii in blood donors. Its specific objectives include: 1. Perform serological screening for anti-T. gondii antibodies (IgM and IgG) in suitable candidates to blood donation and identify the profiles [(non-reactive IgM, IgG non-reactive) (IgM, IgG non-reactive) (IgM, IgG reagent) (IgM nonreactive; IgG reagent)]. 2. Verify the presence of genomic DNA from T. gondii in peripheral blood of donors with the use of gene amplification methods (PCR and conventional real-time PCR). 3. Characterize the donors with positive PCR, the genotypes of T. gondii with the use of molecular markers. Material and methods. Will be selected 700 blood donors suitable for donation at the Blood Center of Sao Jose do Rio Preto, São Paulo State. For each donor will fill a form 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). Samples containing positive by PCR (both) are genotyped with the use of markers SAG1, SAG2 (50-and 30-SAG2, alt.SAG2) SAG3, BTUB, GRA6, C22-8, c29-2, L358, and PK1 apico. As the serological profile obtained from the ELISA, blood donors are separated into four groups (G1, G2, G3 and G4). The G1 will be included donors with IgM and IgG serological profile were negative. In G2, it will include those with IgM and IgG reagent, in G3, those reactive IgM and IgG non-reactive, and G4 non-reactive IgM and IgG reagent. All these groups will be compared with the results of PCR for T. gondii. The results and PCR are compared with each other by X2 or Fisher's exact test. You'll also calculated the odds Ratio. Expected results. It is expected that the results to be achieved with this design contribute to meet the T. gondii circulates in the peripheral blood of donors and allow risk estimate that the presence of such parasites offers patients requiring transfusion of blood products.