Plasmodium falciparum African PfCRT Mutant Isoforms Conducive to Piperaquine Resistance are Infrequent and Impart a Major Fitness Cost

Piperaquine, used in combination with dihydroartemisinin, has been identified as a promising partner drug for uncomplicated treatment and chemoprevention of Plasmodium falciparum malaria in Africa. In light of the earlier spread of piperaquine resistance in Southeast Asia, mediated primarily by mutations in the drug efflux transporter PfCRT, we have explored whether PfCRT mutations would represent a probable path to piperaquine resistance becoming established in Africa. We edited PfCRT mutations known to mediate piperaquine resistance in Southeast Asia into P. falciparum asexual blood stage parasites expressing three prevalent African mutant PfCRT haplotypes. Gene-edited clones were profiled in antimalarial concentration-response and competitive fitness assays. pfcrt-edited parasites expressing the contemporary Southeast Asian T93S or I218F mutations added to the GB4 and Cam783 haplotypes common in Africa did not mediate piperaquine resistance, with partial survival only at low drug concentrations. In contrast, parasites expressing these mutations on the rare PfCRT FCB haplotype, observed mostly in North-East Africa, acquired a moderate level of piperaquine resistance. Dd2GB4, Dd2Cam783, and Dd2FCB lines edited to express the T93S or I218F mutations showed increased susceptibility to chloroquine. Piperaquine-resistant African PfCRT isoforms conferred a substantial fitness cost, manifesting as reduced asexual blood stage parasite growth rates. These findings suggest that piperaquine-resistant PfCRT mutations that emerged in Southeast Asia mediate resistance only in a limited subset of African PfCRT haplotypes, with fitness costs that we suspect would likely preclude dissemination in high-transmission malaria-endemic African regions.