For decades, Southeast Asia (SEA) has been ground zero for the evolution of drug-resistant Plasmodium falciparum malaria. After spawning generations of parasites resistant to chloroquine, sulfadoxine-pyrimethamine, quinine, and mefloquine, this region has now given rise to parasites resistant to artemisinins – the world's frontline antimalarial drugs. These include artemisinin and its derivatives artesunate and artemether, all of which are metabolized to the active compound dihydroartemisinin (DHA) in vivo. Parenteral artesunate has been highly efficacious in reducing malaria morbidity and mortality in SEA  and Africa . Artemisinin combination therapies (ACTs) – oral coformulations of a potent, short-acting artemisinin and a less-potent, long-acting partner drug – have effectively reduced the world's malaria burden, but now face the clear and present danger of artemisinin resistance . This is because higher numbers of parasites that survive exposure to the artemisinin component are now exposed to the partner drug alone. This larger parasite biomass is, thus, more likely to develop partner-drug resistance, which is readily defined by a triad of findings following directly-observed treatment with a high-quality ACT: recrudescent parasitemia within 28–42 days (depending on the ACT), identified by expert microscopic examination of weekly blood smears; adequate partner drug exposure, confirmed by measurement of drug plasma concentrations on day 7; and decreased in-vitro susceptibility of recrudescent parasites to the partner drug, detected by an increase in its median inhibitory concentration (IC50).
In contrast, artemisinin resistance has been more challenging to define, mostly because artemisinins act potently and rapidly to clear parasites from the bloodstream by a unique mechanism involving the spleen [4,5]. Artemisinins are considered prodrugs that are activated by heme iron-mediated cleavage of their endoperoxide moiety within the parasite, a process that forms reactive oxygen intermediates that target nucleophilic groups in parasite proteins and lipids. In patients with P. falciparum infection, this killing process can only be observed in the blood wherein intraerythrocytic ring-stage parasites develop and circulate for 24 h before they sequester in microvessels. When these ring stages are exposed to artemisinins, they consolidate into pyknotic forms resembling Howell–Jolly body inclusions that are efficiently cleared from the bloodstream by ‘pitting,’ a process whereby parasites are squeezed out of their host red blood cells (RBCs) as they pass through tight endothelial slits in the spleen, and the resultant ‘once-infected’ RBCs return intact to the peripheral blood. In patients treated with artemisinins, artemisinin-sensitive parasites rapidly undergo pyknosis and pitting, and, thus, show fast parasite clearance rates. These rates are measured by making frequent parasite counts until parasites are undetectable, log-transforming these counts and plotting them against time, identifying the linear portion of the resultant parasite clearance curve  using a ‘Parasite Clearance Estimator’ tool (http://www.wwarn.org/tools-resources/toolkit/analyse/parasite-clearance-estimator-pce) , and then calculating the parasite clearance half-life (in hours) from the slope of this line.
Artemisinin resistance was first reported from Pailin Province, Western Cambodia, as a slow parasite clearance rate in 2009 . Since then, this clinical phenotype has been documented elsewhere in Cambodia [9,10▪▪], Vietnam [10▪▪,11,12], Thailand [10▪▪,13], Myanmar [10▪▪,14], and China [15▪]. Here, I review a recent series of clinical, epidemiological, genomics, and in-vitro studies that has rapidly transformed our understanding of artemisinin resistance in the human and parasite populations of these Southeast Asian countries.
WHAT IS THE DEFINITION OF ARTEMISININ RESISTANCE?
In Southeast Asian patients with uncomplicated P. falciparum malaria and a starting parasite count at least 10 000 parasites per μl of blood, artemisinin resistance is defined as a parasite clearance half-life at least 5 h following treatment with artesunate monotherapy or an ACT (http://www.who.int/malaria/publications/atoz/status_rep_artemisinin_resistance_sep2014.pdf?ua=1). This 5-h cutoff value reflects the upper limit of parasite clearance half-lives in areas without artemisinin-resistant parasites [10▪▪]. Importantly, parasite clearance half-lives in SEA are not significantly modified by age ; hemoglobin E, a polymorphism carried by 50% of Cambodians ; starting parasite density [9,13]; or relatively lower drug exposures, that is, parasite clearance half-lives were similar in patients randomized to receive 2 or 4 mg/kg artesunate [10▪▪]. Although immunity likely plays a role in parasite clearance in SEA, this has not yet been adequately studied, largely because age is a poor surrogate of adaptive immunity and no in-vitro correlate of parasite-clearing immunity has been established in this region.
To investigate whether adaptive immunity accelerates parasite clearance, two recent studies were conducted in a Malian village where artemisinin resistance is absent and age-dependent reductions in both malaria incidence and parasite density were clearly demonstrated . In the first study , parasite clearance half-lives decreased significantly as age increased, suggesting that age-dependent immunity is involved in clearing ring-infected RBCs within hours of artesunate exposure. In the second study [18▪], younger children cleared their parasites mostly by pitting, suggesting they lacked immune responses that rapidly clear ring-infected RBCs, whereas older children cleared their parasites mostly by a nonpitting, artemisinin-independent mechanism, suggesting they possessed such immune responses. As parasite clearance half-lives are likely influenced by immunity in many areas of Africa, site-specific, age-stratified data are needed to define baseline cutoff values for suspected artemisinin resistance in the future. In areas of Africa where malaria is being eliminated, the progressive loss of immunity may cause a lengthening of parasite clearance half-lives over time, which would not necessarily herald emerging artemisinin resistance.
Artemisinin resistance in P. falciparum has also been defined as a parasite survival rate at least 1% in the ring-stage survival assay (RSA0–3 h) in vitro . In this assay, clinical parasite isolates are adapted to culture, synchronized at the early-ring stage (0–3 h postinvasion of RBCs), exposed to a pharmacologically-relevant dose of DHA for 6 h, and then cultured for an additional 66 h. The percentage of parasites surviving DHA exposure is then calculated as the ratio of parasites surviving exposure to DHA versus those surviving exposure to dimethyl sulfoxide (the DHA solvent). This assay discriminates two groups of parasites, one with less than 1% survival and another with at least 1% survival, which are generally defined as artemisinin sensitive and artemisinin resistant, respectively [19,20▪▪,21,22▪▪]. Importantly, this assay is unable to discriminate these two groups of parasites at the mid-ring and late-ring stages , suggesting that artemisinin resistance is an early-ring-stage phenotype. This finding may account for some discrepancies between parasite clearance half-lives and parasite survival rates in the RSA0–3 h . For example, parasite isolates that are artemisinin resistant in the RSA0–3 h may clear rapidly in patients if they are circulating as mid-to-late ring stages during the time that parasite clearance is measured.
WHAT ARE THE GENETIC DETERMINANTS OF ARTEMISININ RESISTANCE?
Initial genome-wide association studies of parasite clearance half-life implicated two regions of parasite chromosome 13 in artemisinin resistance [23,24], but the specific genetic determinant(s) remained elusive. In a parallel investigation, Ariey et al. [20▪▪] successfully induced artemisinin resistance in a Tanzanian parasite line by exposing it to increasing doses of artemisinin in vitro. By comparing the whole-genome sequences of drug-selected and unselected parasite lines, they identified a single-nucleotide polymorphism (SNP) in the PF3D7_1343700 gene on chromosome 13, which encodes a M476I substitution in the propeller domain of a kelch protein. When compared with a known mammalian ortholog Keap1, the parasite kelch protein (‘K13’) consists of Plasmodium-specific sequences, a bric-à-brac, tramtrack, broad-complex/poxvirus zinc fingers (BTB–POZ) domain, and a six-blade propeller domain (Fig. 1). Validation of K13-propeller polymorphism as a molecular marker of artemisinin resistance in Cambodia was achieved by showing that 17 different K13 mutations were present in parasites from this country (with each parasite clone carrying only one mutation); that the predominant C580Y mutation had rapidly increased in prevalence in areas of Western Cambodia where artemisinin resistance had become common; and that the C580Y, Y493H, and R539T mutations were associated with long parasite clearance half-lives and elevated RSA0–3 h survival rates.
Multiple groups have since made rapid progress in demonstrating K13-propeller polymorphism as a marker of artemisinin resistance elsewhere in SEA, including Vietnam, Thailand, Myanmar, and China by associating the same and additional K13-propeller mutations with slow parasite clearance [10▪▪,15▪,25,26▪]. Molecular surveillance studies have greatly expanded the map of K13-propeller polymorphism to include additional areas of Cambodia , Thailand , Myanmar [29▪,30], China [30,31], and Bangladesh ; some of these mutations have been previously associated with slow parasite clearance at other sites, but most have not and require validation. Currently, C580Y predominates in Cambodia [20▪▪,26▪,27,33,34▪] and along the Thailand–Myanmar border [25,26▪,29▪], whereas F446I predominates along the Myanmar–China border [15▪,29▪,31]. At present, it is unclear how C580Y is approaching fixation in Western Cambodia given that this mutation does not seem to confer higher RSA0–3 h survival rates than other prevalent mutations (e.g., R539T and R543T) [20▪▪,21,22▪▪]. Multiple studies in Africa have detected dozens of K13-propeller mutations – many of which have not yet been observed in SEA – at very low frequency in 17 countries [35▪,36–40] (http://biorxiv.org/content/early/2015/05/22/019737). Whether these K13-propeller mutations cause artemisinin resistance in patients and in vitro also awaits further investigation. Table 1 [10▪▪,15▪,20▪▪,21,22▪▪,25,26▪,27,28,29▪,30–32,34▪,35▪,36–40] (http://biorxiv.org/content/early/2015/05/22/019737) lists all K13-propeller mutations discovered to date, according to their geographic location and association with artemisinin resistance.
In population genetics studies of artemisinin resistance, several surprising and unprecedented findings were made [41,42▪▪]. First, multiple parasite ‘founder’ populations were identified in Cambodia and Vietnam. These groups of highly-differentiated, clonal subpopulations are as different from each other as each of them is to African parasites, suggesting they have undergone extreme recent bottlenecking and subsequent expansion. Second, seven of the 11 founders were found to be artemisinin resistant in patients [41,42▪▪], and three of them were additionally confirmed to be artemisinin resistant in the RSA0–3 h , suggesting that most of them were naturally selected by artemisinins. Third, each founder was tagged by a single K13-propeller mutation, with the C580Y mutation independently emerging on three different founders in Cambodia. Fourth, all seven artemisinin-resistant founders share a common genetic background comprised of four SNPs in genes encoding apicoplast ribosomal protein s10 (arps10 V127 M), ferredoxin (fd D193Y), multidrug resistance 2 transporter (mdr2 T484I), and chloroquine resistance transporter (crt N326S) [42▪▪]. The roles of these mutations in the natural selection of these founders are unknown, but are likely to include increases in fitness. Some possibilities are that they compensate for putative deleterious effects of K13-propeller mutations; potentiate resistance to artemisinins; mediate resistance to previously used drugs (i.e., chloroquine, sulfadoxine-pyrimethamine, quinine, and doxycycline) or currently used ACT partner drugs (i.e., mefloquine, piperaquine, and lumefantrine); or increase parasite transmission to Anopheles mosquito vectors.
WHAT IS THE MOLECULAR MECHANISM OF ARTEMISININ RESISTANCE?
As mammalian kelch proteins can detect oxidants and other stressors, K13-propeller mutations were reasonably implicated in mediating resistance to artemisinin [20▪▪], a prooxidant drug. In one hypothetical model of artemisinin sensitivity and resistance (Fig. 2a), wildtype K13 binds a putative transcription factor and delivers it to ubiquitin ligase, which targets it for proteosomal degradation. When wildtype K13 senses oxidants like artemisinins, it undergoes a conformational change to liberate the transcription factor, which then upregulates the expression of genes involved in counteracting oxidative damage. In this model, the response of wildtype parasites is believed to be too little too late, such that the action of artemisinins is too potent and too rapid for parasites to successfully overcome. In artemisinin-resistant parasites, on the contrary, K13-propeller mutations destabilize the K13-transcription factor interaction, leading to constitutive activation of transcriptional changes that ‘prime’ the parasite to withstand oxidative damage caused by artemisinins.
Given the logical assumption that K13-propeller mutations mediate artemisinin resistance, Straimer et al. [22▪▪] tested this hypothesis directly by using zing-finger nuclease technology to edit the K13 locus in contemporary Cambodian parasite isolates. When three different K13-propeller mutations (C580Y, R539T, and I543T) were edited to the wildtype sequence, the artemisinin-resistance phenotype – as measured in the RSA0–3 h – was completely lost. They also showed that the introduction of five different K13-propeller mutations confer increasing levels of resistance to the Indochinese Dd2 parasite line (Y493H<C580Y<M476I<R539T<I543T), and that introduction of the C580Y mutation confers higher levels of resistance to contemporary parasite isolates from Cambodia than to older parasite lines from Indochina. These data provide compelling evidence that different K13-propeller mutations mediate different levels of artemisinin resistance, and that the level of resistance can be influenced by parasite genetic background. Evidence that C580Y confers artemisinin resistance to the African NF54 parasite line has also been reported .
Although these studies established that K13 mutations confer artemisinin resistance to a variety of parasite clinical isolates and laboratory lines, additional studies were needed to further define the molecular mechanism. In a large population transcriptomics study of P. falciparum isolates obtained directly from Southeast Asian patients with malaria [44▪▪], Mok et al. first identified a subset of parasites that was collected at the early-ring stage of development, that is, when the artemisinin-resistance phenotype is expressed. In analyzing the transcriptional profiles of these isolates against a wide range of corresponding half-lives, these investigators found that artemisinin resistance is associated with increased expression of an ‘unfolded protein response’ pathway involving two major chaperone complexes: Plasmodium reactive oxidative stress complex (PROSC) and TCP-1 ring complex (TRiC). Artemisinin-resistant clinical isolates also showed transcriptional evidence of delayed progression through the intraerythrocytic lifecycle upon cultivation ex vivo. These two transcriptional phenotypes are closely linked to K13-propeller mutations, and may enable parasites to survive artemisinin by first repairing their oxidatively-damaged proteins before progressing through their cell cycle. Future work is needed to integrate these findings with those of two more-recent studies that describe an enhanced cell-stress response  and altered patterns of development  in artemisinin-resistant parasites.
Further progress in exploring mechanisms of artemisinin sensitivity and resistance was recently provided by Mbengue et al. [47▪▪], who report evidence that artemisinins target the sole P. falciparum phosphatidylinositol-3-kinase (PI3K), and that PI3K is the putative binding partner of K13. In their model of artemisinin sensitivity (Fig. 2b), wildtype K13 binds PI3K and delivers it to ubiquitin ligase, which polyubiquitinates K13 and marks it for proteosomal degradation. As these parasites have low basal levels of phosphatidylinositol-3-phosphate (PI3P), the product of PI3K activity, they are highly sensitive to artemisinins, which inhibit PI3K and, thus, prevent the increase in PI3P levels that is presumably needed for parasite growth (PI3P levels normally increase as parasites develop from rings to schizonts). In their corresponding model of artemisinin resistance, mutant K13 fails to bind PI3K. PI3K, thus, avoids being degraded, resulting in high basal levels of PI3K and its product PI3P. As resistant parasites already have high basal levels of PI3P when exposed to artemisinin, they can better withstand the PI3K-inhibiting effects of this drug and, thus, continue their PI3P-dependent growth. How elevated levels of PI3P might mediate artemisinin resistance is not known, but one possibility is that PI3P is involved in membrane biogenesis and fusion events required for parasite growth. Future work is needed to integrate these findings with those of the aforementioned population transcriptomics study [44▪▪], which found no association between PI3K transcript levels and either parasite clearance half-lives or K13-propeller mutations, and to reconcile two very disparate artemisinin modes of action: namely, nonspecific oxidation of multiple parasite proteins versus specific inhibition of PI3K.
WHAT IS THE CLINICAL IMPACT OF ARTEMISININ RESISTANCE?
It is important to emphasize that ACTs still cure patients with slow parasite clearance, provided that the partner drug remains effective. However, slow parasite clearance in ACT-treated patients causes more parasites to be exposed to partner drugs alone, increasing their chance of developing resistance to these drugs and causing ACT failures. As predicted, DHA-piperaquine is now failing to cure malaria in Western Cambodia, where artemisinin resistance is most entrenched. Compared with earlier studies that documented 98% DHA-piperaquine efficacy, three recent studies have reported reduced efficacy in this region. In the first study , efficacy in Pailin and Pursat Provinces was 75 and 89% in 2008–2010. In the second study , in which data were pooled by region, efficacy was 85% in four Western Cambodian provinces (where artemisinin resistance is common) compared with 98% in four eastern Cambodian provinces (where artemisinin resistance is uncommon) in 2011–2013. In this study, the most significant risk factor for treatment failure was the presence of a resistance-associated K13-propeller mutation. In the third study [34▪], efficacy in Oddar Meancheay Province was 46% in 2012–2014. In this study, a significant risk factor for treatment failure was the presence of the K13 C580Y mutation and two other SNPs on chromosomes 10 and 13 that were previously associated with slow parasite clearance . Surprisingly, all three studies were unable to associate treatment failures with elevated piperaquine IC50 values in vitro. As high ACT failure rates in SEA have only been observed in areas where resistance to the partner drug exists, it is likely that piperaquine resistance has indeed emerged. Although this possibility is further suggested by increasing piperaquine IC50 values within multiple study sites over time (unpublished data; ), more robust evidence of piperaquine resistance is needed to identify its genetic markers, elucidate its molecular mechanism, and discover new drugs that circumvent it. Meanwhile, artesunate-mefloquine may be an effective treatment for DHA-piperaquine failures, as suggested by a contemporaneous reduction in mefloquine IC50 values and disappearance of the multicopy pfmdr1 genotype – a molecular marker of mefloquine resistance [33,49,50].
The aggressive global use of ACTs was expected to weaken malaria's stranglehold on the health and economies of the world's most impoverished communities. Unfortunately, the eventual spread of artemisinin resistance from SEA, where ACTs have begun to fail, to Africa, where the world's greatest malaria transmission, morbidity, and mortality occur, seems likely. Multiple international collaborations have defined in-vivo and in-vitro correlates of artemisinin resistance, identified its causal genetic determinant, begun to elucidate its molecular mechanism, and assessed its clinical impact. These collaborative efforts should now be extended to monitor ACT efficacy in areas where K13-propeller mutations are prevalent, test whether currently available drugs cure ACT failures, and advance newly-developed antimalarial compounds into clinical trials.
I thank Socheat Duong, Arjen Dondorp, Nick White, Nick Day, Joel Tarning, Olivo Miotto, Dominic Kwiatkowski, Frédéric Ariey, Didier Ménard, David Fidock, Zbynek Bozdech, Mahamadou Diakité, Pierre Buffet, and Michael Fay for many years of transparent, productive, and enjoyable collaborations; and Chanaki Amaratunga, Seila Suon, Sokunthea Sreng, Pharath Lim, Tatiana Lopera-Mesa, Jennifer Anderson, Dick Sakai, Robert Gwadz, and Thomas Wellems for their efforts in supporting our field studies in Cambodia and Mali.
Financial support and sponsorship
I am funded by the Intramural Research Program of the NIAID, NIH.
Conflicts of interest
There are no conflicts of interest.
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