The pattern of gene amplification of members of the Plasmodium vivax erythrocyte binding-like proteins family across the Amazon rainforest

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, though its function in erythrocyte invasion remains unclear. While copy number variation (CNV) in EBL genes have been well-documented, data on dbp and ebp2 amplifications in P. vivax isolates from South America remain scarce. This study investigates CNV in these two ebl genes across Amazon populations and examines their association with host DARC genotypes. Methods A total of 192 P. vivax isolates from three malaria-endemic sites of the Amazon region (eastern, western, and southwestern) were analyzed. DARC polymorphisms 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.