Background: The malaria elimination plan of the Greater Mekong Subregion (GMS) is jeopardized by the increasing number of Plasmodium vivax infections and emergence of parasite strains with reduced susceptibility to the frontline drug treatment chloroquine/primaquine. This study aimed to determine the evolution of the P. vivax multidrug resistance 1 (Pvmdr1) gene in P. vivax parasites isolated from the China–Myanmar border area during the major phase of elimination. Methods: Clinical isolates were collected from 275 P. vivax patients in 2008, 2012–2013 and 2015 in the China–Myanmar border area and from 55 patients in central China. Comparison was made with parasites from three border regions of Thailand. Results: Overall, genetic diversity of the Pvmdr1 was relatively high in all border regions, and over the seven years in the China–Myanmar border, though slight temporal fluctuation was observed. Single nucleotide polymorphisms previously implicated in reduced chloroquine sensitivity were detected. In particular, M908L approached fixation in the China–Myanmar border area. The Y976F mutation sharply decreased from 18.5% in 2008 to 1.5% in 2012–2013 and disappeared in 2015, whereas F1076L steadily increased from 33.3% in 2008 to 77.8% in 2015. While neutrality tests suggested the action of purifying selection on the pvmdr1 gene, several likelihood-based algorithms detected positive as well as purifying selections operating on specific amino acids including M908L, T958M and F1076L. Fixation and selection of the nonsynonymous mutations are differently distributed across the three border regions and central China. Comparison with the global P. vivax populations clearly indicated clustering of haplotypes according to geographic locations. It is noteworthy that the temperate-zone parasites from central China were completely separated from the parasites from other parts of the GMS. Conclusions: This study showed that P. vivax populations in the China–Myanmar border has experienced major changes in the Pvmdr1 residues proposed to be associated with chloroquine resistance, suggesting that drug selection may play an important role in the evolution of this gene in the parasite populations.

: In the course of malaria elimination in the Greater Mekong Subregion (GMS), malaria epidemiology has experienced drastic spatiotemporal changes with residual transmission concentrated along international borders and the rising predominance of Plasmodium vivax. The emergence of Plasmodium falciparum parasites resistant to artemisinin and partner drugs renders artemisinin-based combination therapies less effective while the potential spread of multidrug-resistant parasites elicits concern. Vector behavioral changes and insecticide resistance have reduced the effectiveness of core vector control measures. In recognition of these problems, the Southeast Asian International Center of Excellence for Malaria Research (ICEMR) has been conducting multidisciplinary research to determine how human migration, antimalarial drug resistance, vector behavior, and insecticide resistance sustain malaria transmission at international borders. These efforts allow us to comprehensively understand the ecology of border malaria transmission and develop population genomics tools to identify and track parasite introduction. In addition to employing in vivo, in vitro, and molecular approaches to monitor the emergence and spread of drug-resistant parasites, we also use genomic and genetic methods to reveal novel mechanisms of antimalarial drug resistance of parasites. We also use omics and population genetics approaches to study insecticide resistance in malaria vectors and identify changes in mosquito community structure, vectorial potential, and seasonal dynamics. Collectively, the scientific findings from the ICEMR research activities offer a systematic view of the factors sustaining residual malaria transmission and identify potential solutions to these problems to accelerate malaria elimination in the GMS.

Background/Objectives: Malaria remains a major global health burden, increasingly complicated by resistance to artemisinin-based therapies. Because artemisinin activation depends on heme and porphyrin chemistry, we sought to exploit host red blood cell (RBC) heme metabolism as a therapeutic vulnerability. This study aims to develop and evaluate a host-directed “bait-and-kill” strategy that selectively sensitizes malaria-infected RBCs to artemisinin. Methods: We integrated quantitative proteomics, erythropoiesis transcriptomic analyses, flow cytometry, and in vitro malaria culture assays to characterize heme metabolism in mature RBCs and Plasmodium falciparum-infected RBCs (iRBCs). The heme precursor 5-aminolevulinic acid (ALA) was used to induce porphyrin accumulation, and dihydroartemisinin (DHA) was applied as the killing agent. Drug synergy, porphyrin accumulation, reactive oxygen species (ROS) induction, and parasite survival were assessed, including ring-stage survival assays using artemisinin-resistant clinical isolates. Results: Mature RBCs retain a truncated heme biosynthesis pathway capable of accumulating porphyrin intermediates, while uninfected RBCs are impermeable to ALA. In contrast, iRBCs exhibit increased membrane permeability, allowing selective ALA uptake and porphyrin accumulation. ALA alone did not induce cytotoxicity or ROS, whereas DHA induced ROS and parasite killing. The ALA + DHA combination resulted in synergistic parasite elimination, including complete clearance of artemisinin-resistant P. falciparum isolates from the Greater Mekong Subregion, with no recrudescence observed over three weeks of culture. Evidence supports a predominant role for host-derived heme metabolites in mediating this synergy. Conclusions: The bait-and-kill strategy selectively exploits host RBC heme metabolism to restore and enhance artemisinin efficacy while sparing uninfected cells. Using clinically safe compounds, this host-directed approach provides a promising, resistance-bypassing framework for malaria treatment and combination drug development.

Heme metabolism is central to the biology of malaria parasites and to the mechanism of action of artemisinin-based therapies. Within malaria-infected red blood cells (RBCs), heme-related chemistry arises from multiple nested metabolic sources that function as “Russian dolls”: the truncated heme biosynthetic capacity of the host erythrocyte, the parasite’s own heme synthesis pathway, and host heme released through hemoglobin digestion in the parasite food vacuole. These overlapping metabolic layers create distinct pools of heme that can influence redox balance and drug activation. Recent studies highlight that exogenous 5-aminolevulinic acid (5-ALA) can perturb host heme biosynthesis in infected erythrocytes, potentially increasing intracellular levels of the heme intermediate protoporphyrin IX and sensitizing parasites to oxidative stress. However, the extent to which such metabolic perturbations affect artemisinin susceptibility depends strongly on parasite stage and exposure duration. Here we review the compartmentalized architecture of heme metabolism in malaria-infected RBCs and discuss how these nested vulnerabilities may be exploited for therapeutic intervention.