Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

Abstract

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 ...

Identification of Three Novel Ring Expansion Metabolites of KAE609, a New Spiroindolone Agent for the Treatment of Malaria, in Rats, Dogs, and Humans

“…Drug resistance to widely used artemisinin-based therapies (Carter et al, 2015;Hott et al, 2015) has become an imminent issue since drugresistance strains were identified in Bangladesh, Cambodia, Thailand, Vietnam, Myanmar and Laos (Haque et al, 2013;Na-Bangchang and Karbwang, 2013;Witkowski et al, 2013). Growing global efforts are focusing on finding new cures to combat malaria drug resistance.…”

Genetic markers of artemisinin resistance in Plasmodium spp. parasites

“…Although all stages are susceptible to artemisinin, a small proportion of the least susceptible stages may survive a single pulse of artemisinin in vitro. Figure 1B illustrates some of the findings for Cambodian-origin parasite clones harbouring K13 variants [27]. Most notably, the exquisite susceptibility of wild-type ring-stage trophozoites is ablated by the pfk13 variant, and this fully resistant stage is extended in duration, thus minimising the proportion of the life cycle spent at the more susceptible stages [mature trophozoite (MT) and early schizont (ES)].…”

“…There is very good evidence from laboratory studies of P. falciparum isolates grown in culture that artemisinin susceptibility varies greatly across the cell cycle [26,27]. The patterns as currently understood for wild-type parasites are represented in cartoon form in Figure 1A.…”

“…The second artesunate dose will find relatively vulnerable MT and ES among the resistant survivors, but the third dose again would encounter highly resistant ERT in the resistant cohort ( Figure 2, bottom panel, top row). If the perturbations of cell cycle dynamics described by Hott et al [27] hold in vivo, it may be that resistant rings are present for longer periods during the cell cycle, which may increase the overall survival.…”

“…All stages are susceptible to artemisinin killing; ERT briefly exhibits exquisite susceptibility, while LRT is the least susceptible stage. (B) Slow-clearing parasite line of Cambodian origin, exemplified by ARC08-88 (clone 4G) described by Hott et al [27]. It is notable that other artemisinin-tolerant clones described by these authors displayed a shortened in vitro life cycle of under 40 h. Abbreviations: DHA: dihydroartemisinin; h: hours post-invasion in a synchronised ring-stage culture; full susc: development stage is fully susceptible to a 3-4 h pulse of 700 nM DHA, and no further growth is detected after the drug pulse; part susc: partly susceptible to the 700 nM DHA pulse, such that some continued growth occurs after the drug is washed away.…”