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ABSTRACTCurrent efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparumresistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates.IMPORTANCEThe global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite Plasmodium. Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparumresistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparumchloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance.

ABSTRACTWhole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivoculture system. Current techniques used to enrich P. vivaxDNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivaxgDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivaxcore genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivaxsequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivaxisolates from clinical samples that can be applied to other neglected microbial pathogens.IMPORTANCEMalaria is a disease caused by Plasmodiumparasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivaxis the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodiumparasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivaxis challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivaxgenomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.