Abstract / Summary
Malaria remains a major global health challenge, with Plasmodium vivax as a key contributor to disease burden across endemic regions. Apical membrane antigen-1 ( PvAMA1 ) and erythrocyte binding protein ( PvEBP ) of P. vivax are the genes encoding erythrocyte invasion protein which promising as a target of erythrocytic vaccine candidate. However, limited genetic study of PvAMA1 and PvEBP in Pakistan was revealed. The objective was to investigates the genetic diversity of PvAMA-1 and PvEBP gene in isolates obtained from diagnostic facilities in malaria-endemic regions of Pakistan. A total of 50 leftover blood samples collecting from diagnostic facilities located in Khyber Pakhthukhwa (KP) and Sindh, Pakistan between May and October 2025 were analyzed by polymerase chain reaction (PCR) and sequencing. Genetic polymorphisms, genetic diversity, neutral selection, population differentiation and haplotype network were performed. Twenty-six and 25 non-synonymous mutations were revealed in PvAMA1 and PvEBP , respectively. Considering haplotypes, 28 haplotypes of PvAMA1 and 25 haplotypes of PvEBP were identified. Notably, one previously unreported non-synonymous mutation of PvAMA1, while 11 non-synonymous mutations of PvEBP were observed. The full-length sequence of PvAMA1 revealed a slightly higher nucleotide diversity (π = 0.00784 ± 0.00056) with the highest diversity located in domain I (π = 0.01973 ± 0.00066). Lower nucleotide diversity was identified in PvEBP (π = 0.00180 ± 0.00013). The neutrality test, Fu and Li’s D* and F* was statistically significant positive in domain I of PvAMA1 ( p < 0.02) with positive values suggesting a significant departure from neutral expectations occur in this region. Global population differentiation analysis by F ST revealed low to high differentiation of both PvAMA1 and PvEBP. PvAMA1 exhibited higher genetic diversity than PvEBP among the Pakistani isolates, with the observed patterns broadly consistent with those reported in global P. vivax populations. The observed genetic diversity of PvAMA1 and PvEBP provides baseline information on sequence variation in these antigen genes among the analyzed Pakistani isolates.