Bower-Lepts, C.; Jangra, A.; Kanatani, S.; Tripathi, A.; Mlambo, G.; McCotter-Gonzalez, A.; Romano, L.; Orena, S.; Okitwi, M.; Niare, K.; Rosenthal, P. J.; Conrad, M.; Sinnis, P.; Mok, S.

Effective control of falciparum malaria depends on the sustained efficacy of frontline antimalarial drugs, particularly artemether-lumefantrine (AL), the most widely used therapy in Africa. However, the emergence of artemisinin partial resistance and reduced lumefantrine susceptibility in eastern Africa threaten malaria control and elimination. Robust genetic markers of decreased susceptibility to lumefantrine remain elusive, and our understanding of artemisinin resistance is incomplete. We report results of a Plasmodium falciparum genetic cross between a drug-sensitive line and a Ugandan strain exhibiting reduced susceptibility to dihydroartemisinin and lumefantrine. Targeted deep sequencing of progeny pools and 460 recombinant progeny clones derived under drug pressures revealed distinct haplotypic signatures. Drug-selection experiments identified genetic polymorphisms in Plasmodium falciparum px1, encoding a phosphoinositide-binding protein, as the strongest correlates of reduced susceptibility to dihydroartemisinin and lumefantrine. The PX1 PIN haplotype (L1222P, M1701I, D1705N) recently discovered in Ugandan parasites was highly enriched following dihydroartemisinin or lumefantrine treatment of pooled mixtures of genetically diverse Ugandan clinical isolates. This haplotype was associated with reduced susceptibility to dihydroartemisinin and lumefantrine, compared to wild-type sequence, in culture-adapted Ugandan P. falciparum lines. These results confirm that PX1 mutations were selected across geographically distinct Ugandan parasite backgrounds. Long-term competitive fitness assays demonstrated that PX1 mutations confer asexual blood-stage parasites with a growth advantage, potentially explaining a rapid rise of PX1 PIN alleles over the last two decades in Uganda. Overall, our data suggest the PX1 PIN haplotype is a robust marker of reduced AL susceptibility in African P. falciparum, enabling surveillance of emerging drug resistance.