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Plasmodium falciparum and Plasmodium vivax, the two protozoan parasite species that cause the majority of cases of human malaria, have developed resistance to nearly all known antimalarials. The ability of malaria parasites to develop resistance is primarily due to the high numbers of parasites in the infected person's bloodstream during the asexual blood stage of infection in conjunction with the mutability of their genomes. Identifying the genetic mutations that mediate antimalarial resistance has deepened our understanding of how the parasites evade our treatments and reveals molecular markers that can be used to track the emergence of resistance in clinical samples. In this review, we examine known genetic mutations that lead to resistance to the major classes of antimalarial medications: the 4-aminoquinolines (chloroquine, amodiaquine and piperaquine), antifolate drugs, aryl amino-alcohols (quinine, lumefantrine and mefloquine), artemisinin compounds, antibiotics (clindamycin and doxycycline) and a napthoquinone (atovaquone). We discuss how the evolution of antimalarial resistance informs strategies to design the next generation of antimalarial therapies., (© The Author(s) 2019. Published by Oxford University Press.)

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance 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 (IC 90 ) or 50% parasite killing (50% lethal dose [LD 50 ]). 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 pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates. IMPORTANCE The 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 falciparum resistance 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 falciparum chloroquine 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., (Copyright © 2017 Dhingra et al.)

ImportanceMalaria is a devastating disease caused by Plasmodium parasites. The evolution of parasite drug resistance continues to hamper progress toward malaria elimination, and despite extensive efforts to control malaria, it remains a leading cause of death in Mozambique and other countries in the region. The development of successful vaccines and identification of molecular markers to track drug efficacy are essential for managing the disease burden. We present an analysis of the parasite genome in Mozambique, a country with one of the highest malaria burdens globally and limited available genomic data, revealing current selection pressure. We contribute additional evidence to limited prior studies supporting the effectiveness of SWGA in producing reliable genomic data from complex clinical samples. Our results provide the identity of genomic loci that may be associated with current antimalarial drug use, including artemisinin and lumefantrine, and reveal selection pressure predicted to compromise the efficacy of current vaccine candidates.