Prevalence and genetic diversity of simian malaria in wild macaque populations across Thailand: Implications for human health

Over the past year, P. falciparum infections have declined in Thailand, yet nonhuman primate malaria infections have correspondingly increased, including Plasmodium knowlesi and P. cynomolgi . Nevertheless, little is known about simian malaria in its natural macaque hosts, Macaca mulatta and Macaca fascicularis . This study aims to address several research questions, including the prevalence and distribution of simian malaria in these two Thai wild macaque species, variations in infection between different macaque species and between M. fascicularis subspecies, and the genetic composition of these pathogens. Blood samples were collected from 82 M. mulatta and 690 M. fascicularis across 15 locations in Thailand, as well as two locations in Vietnam and Myanmar. We employed quantitative real -time PCR targeting the Plasmodium genus-specific 18S ribosomal RNA (rRNA) gene to detect malaria infection, with a limit of detection set at 1,215.98 parasites per mL. We genotyped eight microsatellite markers, and the P. cynomolgi dihydrofolate reductase gene (DHFR) was sequenced ( N = 29). In total, 100 of 772 samples (13 %) tested positive for malaria, including 45 (13 %) for P. cynomolgi , 37 (13 %) for P. inui , 16 (5 %) for P. coatneyi , and 2 (0.25 %) for Hepatocystis sp . in Saraburi, central and Ranong, southern Thailand. Notably, simian malaria infection was observed exclusively in M. fascicularis and not in M. mulatta ( P = 0.0002). Particularly, P. cynomolgi was detected in 21.7 % (45/207) of M. f. fascicularis living in Wat Tham Phrapothisat, Saraburi Province. The infection with simian malaria was statistically different between M. fascicularis and M. mulatta ( P = 0.0002) but not within M. fascicularis subspecies ( P = 0.78). A haplotype network analysis revealed that P. cynomolgi shares a lineage with reference strains obtained from macaques. No mutation in the predicted binding pocket of PcyDHFR to pyrimethamine was observed. This study reveals a significant prevalence of simian malaria infection in M. fascicularis . The clonal genotypes of P. cynomolgi suggest in-reservoir breeding. These findings raise concerns about the potential spread of nonhuman primate malaria to humans and underscore the need for preventive measures.