Evidence for the early emergence of piperaquine-resistant Plasmodium falciparum malaria and modeling strategies to mitigate resistance

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter's central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy. Author summary The recent emergence of Plasmodium falciparum parasite resistance to the antimalarial drug piperaquine (PPQ) has contributed to frequent treatment failures across Southeast Asia, originating in Cambodia. Here, we show that earlier reports of PPQ resistance in Yunnan Province, China could be explained by the unique China C variant of the P. falciparum chloroquine resistance transporter PfCRT. Gene-edited parasites show a loss of fitness and parasite resensitization to the chemically related former first-line antimalarial chloroquine, while acquiring PPQ resistance via drug efflux. Molecular features of drug resistance were examined using biochemical assays to measure mutant PfCRT-mediated drug transport and molecular dynamics simulations with the recently solved PfCRT structure to assess changes in the central drug-binding cavity. We also describe a new computational model that incorporates parasite mutation rates, fitness costs, antimalarial susceptibilities, and drug pharmacological profiles to predict how infections with parasite strains expressing distinct PfCRT variants can evolve and be selected in response to different drug pressures and regimens. Simulations predict that a three-day regimen of PPQ plus chloroquine would be fully effective at preventing recrudescence of drug-resistant infections.