Plasmepsin II – III Copy Number Variation Accounts For Bimodal Piperaquine Resistance Among Cambodian Plasmodium Falciparum Isolates With Single Pfmdr1 Copy Paper submitted

Highly drug resistant Plasmodium falciparum parasites in Southeast Asia (SEA) threaten regional malaria elimination, and risk spreading to other malaria endemic areas. These extremely drug resistant malaria parasites harbor both artemisinin (ART) and piperaquine (PPQ) resistant phenotypes, compromising effective use of ART Combination Therapy (ACT) to treat patients and reduce the malaria burden. It is crucial to understand mechanisms of drug resistance among these parasites to: track their emergence and spread; understand the biological processes that mediate this drug resistance; and, ultimately develop alternative antimalarial drugs or strategies that offset or circumvent drug resistance. To this end, we leveraged a culture-adapted set of parasites from Pursat and Pailin, Cambodia collected as part of the Tracking Resistance to Artemisinin Collaboration (TRAC) to investigate ART and PPQ resistance. Exposure of these culture-adapted parasites to PPQ revealed a bimodal dose-response curve where area under the curve (AUC) correlated to PPQ resistance. Genetic analysis revealed that these PPQ resistant parasites exhibit changes in copy number variants (CNVs) for plasmepsin II and plasmepsin III (positive association) and pfmdr1 (negative association). A panel of isogenic subclones with different plasmepsin II – III CNV levels that harbor a single copy of pfmdr1 reinforce the importance of plasmepsin II – III CNV and minimize the impact of pfmdr1 CNV on PPQ resistance. Discordant parasites that exhibit high levels of PPQ resistance without increase in plasmepsin II – III CNV were also evident. These results confirm PPQ resistant parasites in Cambodia, support the importance of plasmepsin II – III CNV over pfmdr1 CNV in mediating PPQ resistance, and suggest additional or alternate genetic loci are important for PPQ resistance.