bArtemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance.A rtemisinin combination therapy (ACT) is the recommended treatment for multidrug-resistant malaria by the World Health Organization. These combinations rely on the fast-acting properties of the artemisinin derivative to rapidly clear parasites with the longer-acting partner drug clearing residual parasites, reducing the frequent observation of recrudescent infections when artemisinin drugs are used alone. Unfortunately, resistance to artemisinin derivatives has emerged in southeast Asia and now threatens the utility of the most important treatment options for Plasmodium falciparum malaria that is resistant to most antimalarial drugs. Clinical resistance to artemisinin is expressed as a reduced rate of parasite clearance from peripheral blood, resulting in a parasite clearance half-life of Ͼ5 h (1). Resistance to artemisinin is a heritable trait, linked to mutations in the Kelch propeller domains of Pf3D7_1343700, and it appears to be spreading in southeast Asia (1-3). Clearly, a public health disaster is looming if artemisinin resistance spreads globally, like the selective sweeps previously observed for chloroquine and pyrimethamine resistance (4-6).Despite the well-characterized phenotype of clinical resistance to artemisinin, resistance phenotypes in classical in vitro drug susceptibility assays, used successfully for other antimalarial drugs, have not been useful for detecting artemisinin resistance (7). Two modified in vitro assays have been used to assess resistance with some success (8, 9), with both assessing reduced susceptibility in the ring stage of development in erythrocytes; however, stable in vitro phenotypes in culture-adapted parasites remain elusive. Given the ...

The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen in P. falciparum to identify genes allowing parasites to survive febrile temperatures. Screening identifies more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants are more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin include highly essential, conserved pathways associated with protein-folding, heat shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the ancestral genome of the parasite’s algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general are upregulated in response to heat shock, as are other Plasmodium genes with orthologs in plant and algal genomes. Plasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiont-derived genes in the parasite’s genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors.

Though malaria mortality rates are down 48% globally since 2000, reported occurrences of resistance against current therapeutics threaten to reverse that progress. Recently, antimalarials that were once considered unsuitable therapeutic agents have been revisited to improve physicochemical properties and efficacy required for selection as a drug candidate. One such compound is 4(1H)-quinolone ICI 56,780, which is known to be a causal prophylactic that also displays blood schizonticidal activity against P. berghei. Rapid induction of parasite resistance, however, stalled its further development. We have completed a full structure-activity relationship study on 4(1H)-quinolones, focusing on the reduction of cross-resistance with atovaquone for activity against the clinical isolates W2 and TM90-C2B, as well as the improvement of microsomal stability. These studies revealed several frontrunner compounds with superb in vivo antimalarial activity. The best compounds were found to be curative with all mice surviving a Plasmodium berghei infection after 30 days.

Artemisinin-resistant P. falciparum have a fitness advantage to survive and predominate in the population even in the face of infrequent exposure to artemisinin drugs.