Abstract
BACKGROUND: In Plasmodium vivax malaria, the Duffy Binding Protein (DBP), a key erythrocyte binding-like (EBL) protein, enables invasion of DARC (Duffy Antigen/Receptor for Chemokines) positive reticulocytes. Another EBL member, the erythrocyte binding protein 2 (EBP2, also known as EBP/ DBP2), shares structural features with DBP, suggesting a role in erythrocyte invasion. P. vivax genomes may harbour multiple ebl gene copies, though their functional role is unclear. Considering DBP and EBP2 as key vaccine candidates, this study investigates copy number variation (CNV) in these two ebl genes across Amazonian populations. Furthermore, the relationship between CNV and host DARC genotypes was investigated, as DARC is the best-characterized host receptor associated with susceptibility or resistance to P. vivax. METHODS: A total of 191 P. vivax isolates from three malaria-endemic sites of the Amazon region (eastern, western, and southwestern) were analysed. DARC polymorphisms associated with functional (FY*A, FY*B) and non-functional (FY*B(ES)) alleles were genotyped by real-time PCR with allele-specific oligonucleotides. CNV at dbp and ebp2 loci was estimated by quantitative PCR (qPCR), using the β-tubulin gene as an internal reference. Gene copy numbers were stratified by geographical origin and host DARC genotype. RESULTS: Amplification of dbp and ebp2 genes varied across Amazon regions. In the western region, 25% of P. vivax isolates showed dbp gene amplification (up to 8 copies), compared to 2-9% in the southwestern and eastern regions (2-3 copies). Overall, ebp2 amplification was less frequent, detected in 15% of P. vivax isolates from the western and in 1-4% from other regions. In the study areas, all individuals were DARC positives, and no association was observed between gene CNVs and DARC genotypes. CONCLUSIONS: This study identifies dbp and ebp2 gene amplifications in P. vivax isolates from the Amazon rainforest, with regional CNV variation but no association with any DARC-positive genotype. These findings support further investigation into gene amplifications to elucidate their biological and immunological significance in DARC-positive populations.