Molecular analysis of DHFR and DHPS gene mutations in Plasmodium cynomolgi from humans and macaques in Southeast Asia

Plasmodium cynomolgi is an emerging zoonotic malaria parasite in Southeast Asia, infecting both humans and macaques. In this study, we investigated mutations in the DHFR and DHPS genes of P. cynomolgi from humans and macaques, comparing them to known resistance mutations in P. falciparum and P. vivax . We also examined how these mutations affect antifolate drug binding, which may influence treatment efficacy and resistance. Nine asymptomatic human blood samples from Cambodia and 29 macaque samples from Thailand were analyzed. Human samples included eight P. cynomolgi monoinfections and one mixed infection with P. vivax , while all macaque samples were monoinfections. The PcyDHFR and PcyDHPS genes were amplified, sequenced, and subjected to haplotype analysis. Human samples from Battambang, Cambodia were 100% identical to the P. cynomolgi RO strain, showing no DHFR mutations and one DHPS mutation (V451I). In contrast, macaque samples from Saraburi, Thailand showed PcyDHFR mutations N44T and C49S, and two haplotypes based on I7 variation – haplotype 1 (72.41%) with wild-type I7 and haplotype 2 (27.59%) with the I7 mutation. PcyDHPS mutations were identical across macaque isolates. Protein structures of Pcy DHFR and Pcy DHPS were modeled using SWISS-MODEL, focusing on the N- and C-terminals. Mutations occurred near catalytic sites but did not significantly affect binding affinity, based on molecular docking with eight antifolate drugs. These findings suggest that current antifolate drugs remain potentially effective against P. cynomolgi , and highlight the importance of monitoring drug resistance in zoonotic malaria.