Genetic analysis of two recombinant antigens in Trypanosoma cruzi and their possible immunological use to discriminate clinical forms of disease.

SALDAÑA, GUSTAVO; LONGHI SILVIA A; DIAZ ISABEL; CURTO, MARÍA ANGELES; OSUNA ANTONIO; SCHIJMAN ALEJANDRO G

Mar del Plata

Congreso; X Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2014

Sociedad Argentina de Protozoologia

Gold standard diagnosis of chronic Chagas disease (cCD) is based on serological response to antibodies. However, serological kits, mostly those based on recombinant antigens do not depict similar accuracy in different geographic regions. A hypothesis for this is genetic diversity of T.cruzi, whose populations are clustered into six discrete typing units (DTUs) with diverse regional distribution and probably linked to varied clinical forms of chronic phase. Our aim was to characterize two widely used recombinant antigens (JL7 and 1F8) in parasite stocks representing the 6 DTUs. Coding regions were amplified from genomic DNA of six stocks. Primers were designed from CL Brener genome project databank; PCR products were cloned and recombinants were isolated for sequencing and multiple alignments using ClustalT-coffee software. Alignment of 1F8 translated ORFs showed no significant differences among DTUs (90% overall similarity, only 4 conservative substitutions in 198 aa, except for DTUIII with 3 non-conservative changes in the N-term). In contrast, alignment of JL7 ORFs showed significant differences mainly in the C-term region (14 conservative and 12 non-conservative aa substitutions in positions 51-131). The cladogram showed three clusters: Cluster 1 (DTUI and III); cluster 2 (DTUII) and cluster 3 (DTUIV, V, and VI). Accordingly, 18 peptides covering representing the different variants were synthetized to carry out ELISA assays with sera from patients of different regions and clinical manifestations (asymptomatic, cardiac and gastrointestinal). A preliminary assay in sera from DTU I infected patients showed peptides with differential reactivity: JL7-6/10/12 for asymptomatic; JL7-8 for cardiopaths and JL7-11 for gastrointestinal cases (p=0.01 Turkey test). A higher number of serum samples are currently under analysis. Our findings encourage further investigation of these peptides for discrimination of clinical status in cCD patients from different geographic origins.