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.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
1. Dondorp A, Nosten F, Stepniewska K, et al. Artesunate versus quinine for treatment of severe falciparum malaria
: a randomised trial. Lancet 2005; 366:717–725.
2. Dondorp AM, Fanello CI, Hendriksen IC, et al. Artesunate versus quinine in the treatment of severe falciparum malaria
in African children (AQUAMAT): an open-label, randomised trial. Lancet 2010; 376:1647–1657.
3. Dondorp AM, Fairhurst RM, Slutsker L, et al. The threat of artemisinin
. N Engl J Med 2011; 365:1073–1075.
4. Chotivanich K, Udomsangpetch R, Dondorp A, et al. The mechanisms of parasite clearance after antimalarial treatment of Plasmodium falciparum malaria
. J Infect Dis 2000; 182:629–633.
5. Buffet PA, Milon G, Brousse V, et al. Ex vivo perfusion of human spleens maintains clearing and processing functions. Blood 2006; 107:3745–3752.
6. White NJ. The parasite clearance curve. Malar J 2011; 10:278–285.
7. Flegg JA, Guerin PJ, White NJ, Stepniewska K. Standardizing the measurement of parasite clearance in falciparum malaria
: the parasite clearance estimator. Malar J 2011; 10:339–352.
8. Dondorp AM, Nosten F, Yi P, et al. Artemisinin resistance
in Plasmodium falciparum malaria
. N Engl J Med 2009; 361:455–467.
9. Amaratunga C, Sreng S, Suon S, et al. Artemisinin
-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. Lancet Infect Dis 2012; 12:851–858.
10▪▪. Ashley EA, Dhorda M, Fairhurst RM, et al. Spread of artemisinin resistance
in Plasmodium falciparum malaria
. N Engl J Med 2014; 371:411–423.
This study mapped artemisinin resistance (parasite clearance half-life ≥5 h) in multiple sites across SEA, showing that K13-propeller polymorphism is also a molecular marker of this phenotype outside Cambodia. It greatly increased the number of K13-propeller mutations associated with long parasite clearance half-life, and associated K13-mutant, slow-clearing parasites with increased pretreatment and posttreatment gametocytemia, suggesting that artemisinin-resistant parasites have a potential transmission advantage. This study, thus, argues strongly for the use of single low-dose primaquine to kill mature gametocytes and stop the spread of artemisinin-resistant parasites.
11. Thriemer K, Hong NV, Rosanas-Urgell A, et al. Delayed parasite clearance after treatment with dihydroartemisinin-piperaquine in Plasmodium falciparum malaria
patients in central Vietnam. Antimicrob Agents Chemother 2014; 58:7049–7055.
12. Hien TT, Thuy-Nhien NT, Phu NH, et al. In vivo susceptibility of Plasmodium falciparum to artesunate in Binh Phuoc Province, Vietnam. Malar J 2012; 11:355–366.
13. Phyo AP, Nkhoma S, Stepniewska K, et al. Emergence of artemisinin
on the western border of Thailand: a longitudinal study. Lancet 2012; 379:1960–1966.
14. Kyaw MP, Nyunt MH, Chit K, et al. Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar. PLoS One 2013; 8:e57689–e57699.
15▪. Huang F, Takala-Harrison S, Jacob CG, et al. A single mutation in K13 predominates in Southern China and is associated with delayed clearance of Plasmodium falciparum following artemisinin
treatment. J Infect Dis 2015; [Epub ahead of print].
This study associated the K13 F446I mutation with artemisinin resistance in Southern China, along its border with Myanmar. The prevalence of F446I is very high (36.5%) in this region, placing it at risk of ACT failures in the near future.
16. Lopera-Mesa TM, Doumbia S, Konate D, et al. Impact of red blood cell variants on childhood malaria
in Mali: a prospective cohort study. Lancet Haematol 2015; 2:e140–e149.
17. Lopera-Mesa TM, Doumbia S, Chiang S, et al. Plasmodium falciparum clearance rates in response to artesunate in Malian children with malaria
: effect of acquired immunity. J Infect Dis 2013; 207:1655–1663.
18▪. Ndour PA, Lopera-Mesa TM, Diakite SA, et al. Plasmodium falciparum clearance is rapid and pitting independent in immune Malian children treated with artesunate for malaria
. J Infect Dis 2015; 211:290–297.
This study showed that in Mali, where P. falciparum is artemisinin sensitive, older children clear their parasites following artesunate treatment faster than younger children. This clearance is due to a nonpitting, artemisinin-independent mechanism (likely age-dependent adaptive immunity), which removes ring-infected RBCs from circulation and, thus, hastens parasite clearance. Such immunity may forestall the development of ACT resistance in Africa by helping to decrease the parasite biomass exposed to artemisinins and ACT partner drugs.
19. Witkowski B, Amaratunga C, Khim N, et al. Novel phenotypic assays for the detection of artemisinin
-resistant Plasmodium falciparum malaria
in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 2013; 13:1043–1049.
20▪▪. Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin
-resistant Plasmodium falciparum malaria
. Nature 2014; 505:50–55.
This study described K13-propeller polymorphism as a molecular marker of artemisinin resistance in vivo and in vitro by associating several mutations with long parasite clearance half-lives and elevated RSA0-3 h survival rates in Cambodia. Although the C580Y mutation predominated in Western Cambodia, 16 additional mutations were identified in the country, thus indicating that molecular surveillance for artemisinin resistance will require sequencing the entire K13-propeller region. These discoveries enabled the Tracking Resistance to Artemisinin Collaboration and other research groups to rapidly map the contemporary prevalence of this marker throughout Southeast Asia and Africa.
21. Amaratunga C, Witkowski B, Dek D, et al. Plasmodium falciparum founder populations in western Cambodia have reduced artemisinin
sensitivity in vitro. Antimicrob Agents Chemother 2014; 58:4935–4937.
22▪▪. Straimer J, Gnadig NF, Witkowski B, et al. Drug resistance
. K13-propeller mutations confer artemisinin resistance
in Plasmodium falciparum clinical isolates. Science 2015; 347:428–431.
This study showed that K13-propeller polymorphism is not only a marker of artemisinin resistance in SEA, but also causes this phenotype. Using zinc-finger nucleases to edit K13-propeller mutations to the wildtype sequence in parasite clinical isolates from Cambodia, this study established that K13-propeller polymorphism confers artemisinin resistance (increased RSA0-3 h survival rate). The study also showed that different K13-propeller mutations confer different levels of artemisinin resistance to the Indochinese Dd2 parasite line, and that the predominant C580Y mutation confers greater levels of resistance to contemporary clinical isolates than to older laboratory-adapted lines from Indochina. Collectively, these data suggest that the emergence of artemisinin resistance is driven not only by K13-propeller mutations, but also parasite genetic background.
23. Takala-Harrison S, Clark TG, Jacob CG, et al. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin
treatment in Southeast Asia. Proc Natl Acad Sci U S A 2013; 110:240–245.
24. Cheeseman IH, Miller BA, Nair S, et al. A major genome region underlying artemisinin resistance
. Science 2012; 336:79–82.
25. Nyunt MH, Hlaing T, Oo HW, et al. Molecular assessment of artemisinin resistance
markers, polymorphisms in the k13 propeller, and a multidrug-resistance
gene in the eastern and western border areas of Myanmar. Clin Infect Dis 2015; 60:1208–1215.
26▪. Takala-Harrison S, Jacob CG, Arze C, et al. Independent emergence of artemisinin resistance
mutations among Plasmodium falciparum in Southeast Asia. J Infect Dis 2015; 211:670–679.
This study showed that some K13-propeller mutations (e.g., C580Y) have independently emerged in different parasite populations in different regions of SEA, and that parasites do not seem to be spreading from Cambodia to Myanmar. These findings have important implications for the containment of artemisinin resistance in SEA, in that they increase the focus on preventing the local emergence of resistant parasites.
27. Bosman P, Stassijns J, Nackers F, et al. Plasmodium prevalence and artemisinin
-resistant falciparum malaria
in Preah Vihear Province, Cambodia: a cross-sectional population-based study. Malar J 2014; 13:394–403.
28. Talundzic E, Okoth SA, Congpuong K, et al. Selection and spread of artemisinin
-resistant alleles in Thailand prior to the global artemisinin resistance
containment campaign. PLoS Pathog 2015; 11:e1004789–e1004803.
29▪. Tun KM, Imwong M, Lwin KM, et al. Spread of artemisinin
-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker. Lancet Infect Dis 2015; 15:415–421.
This study showed that K13-propeller mutations are highly prevalent in Myanmar, being found in 39% of samples from 55 sites in 10 administrative districts in 2013–2014. This prevalence is even higher (47%) in a region only 25 km from the Myanmar–India border, suggesting the very real possibility that artemisinin-resistant parasites have already spread locally into India.
30. Feng J, Zhou D, Lin Y, et al. Amplification of pfmdr1, pfcrt, pvmdr1, and K13 propeller polymorphisms associated with Plasmodium falciparum and Plasmodium vivax isolates from the China-Myanmar border. Antimicrob Agents Chemother 2015; 59:2554–2559.
31. Wang Z, Shrestha S, Li X, et al. Prevalence of K13-propeller polymorphisms in Plasmodium falciparum from China-Myanmar border in 2007–2012. Malar J 2015; 14:168–174.
32. Mohon AN, Alam MS, Bayih AG, et al. Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013). Malar J 2014; 13:431–437.
33. Leang R, Taylor WR, Bouth DM, et al. Evidence of falciparum malaria
and piperaquine in western Cambodia: dihydroartemisinin-piperaquine open-label multicenter clinical assessment. Antimicrob Agents Chemother 2015; 59:4719–4726.
34▪. Spring MD, Lin JT, Manning JE, et al. Dihydroartemisinin-piperaquine failure associated with a triple mutant including kelch13 C580Y in Cambodia: an observational cohort study. Lancet Infect Dis 2015; 15:683–691.
This study showed that DHA-piperaquine treatment failures are occuring in 54% of patients in Oddar Meanchey Province in 2013–2014, thus confirming ACT resistance in a fifth Western Cambodian province. The presence of the K13-propeller C580Y mutation and two additional SNPs was associated with a 5.4-fold greater risk of treatment failure and higher piperaquine IC50in vitro, linking artemisinin resistance to the evolution of piperaquine resistance.
35▪. Taylor SM, Parobek CM, DeConti DK, et al. Absence of putative artemisinin resistance
mutations among Plasmodium falciparum in Sub-Saharan Africa: a molecular epidemiologic study. J Infect Dis 2015; 211:680–688.
This study showed that multiple K13-propeller mutations are present at low frequency throughout sub-Saharan Africa, and that none of them have been associated with artemisinin resistance in SEA. Given that many of these mutations have not been observed in SEA (where other mutant alleles have been naturally selected to high frequency) and there is limited allele sharing between parasite populations, these data suggest a large reservoir of naturally-occurring K13 variation in Africa from which resistance alleles may be selected in the future.
36. Kamau E, Campino S, Amenga-Etego L, et al. K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa. J Infect Dis 2015; 211:1352–1355.
37. Escobar C, Pateira C, Lobo E, et al. Polymorphisms in Plasmodium falciparum K13-propeller in Angola and Mozambique after the introduction of the ACTs. PLoS One 2015; 10:e0119215–e0119221.
38. Ouattara A, Kone A, Adams M, et al. Polymorphisms in the K13-propeller gene in artemisinin
-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali. Am J Trop Med Hyg 2015; 92:1202–1206.
39. Hawkes M, Conroy AL, Opoka RO, et al. Slow clearance of Plasmodium falciparum in severe pediatric malaria
, Uganda, 2011–2013. Emerg Infect Dis 2015; 21:1237–1239.
40. Conrad MD, Bigira V, Kapisi J, et al. Polymorphisms in K13 and falcipain-2 associated with artemisinin resistance
are not prevalent in Plasmodium falciparum isolated from Ugandan children. PLoS One 2014; 9:e105690–e105696.
41. Miotto O, Almagro-Garcia J, Manske M, et al. Multiple populations of artemisinin
-resistant Plasmodium falciparum in Cambodia. Nat Genet 2013; 45:648–655.
42▪▪. Miotto O, Amato R, Ashley EA, et al. Genetic architecture of artemisinin
-resistant Plasmodium falciparum. Nat Genet 2015; 47:226–234.
This study associates artemisinin-resistant founder populations with the presence of K13-propeller polymorphism and a particular 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). This finding, which was observed both in the Greater Mekong Subregion and along the Thailand–Myanmar border, strongly suggests that K13-propeller mutations by themselves are not naturally selected in the human and mosquito populations of SEA. Instead, it seems that additional SNPs are needed to increase fitness in these geographical settings. The finding that this genetic background is not seen in Africa suggests that the naturally-occurring K13-propeller mutations observed on this continent cannot yet give rise to artemisinin-resistant subpopulations. The genetic background of four SNPs may, therefore, be considered a molecular marker for identifying areas most at risk of artemisinin resistance in the future.
43. Ghorbal M, Gorman M, Macpherson CR, et al. Genome editing in the human malaria
parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 2014; 32:819–821.
44▪▪. Mok S, Ashley EA, Ferreira PE, et al. Drug resistance
. Population transcriptomics of human malaria
parasites reveals the mechanism of artemisinin resistance
. Science 2015; 347:431–435.
This study reports the largest ex-vivo transcriptomics study of any infectious disease. By analyzing the transcriptional profiles of more than 1000 P. falciparum clinical isolates, this study identified parasites at the early-ring stage of parasite development that expressed a transcriptional profile associated with long parasite clearance half-lives and K13-propeller mutations. This profile indicates that parasites, before they have encountered artemisinin in the patient, have already decelerated their growth and upregulated their ‘unfolded protein response’ pathway. Together, these findings suggest that parasites have evolved to anticipate artemisinin exposure, and are, thus, better able to first repair oxidative protein damage before progressing through their lifecycle. The identification of two upregulated chaperone complexes (PROSC and TRiC), with multiple molecular components, has opened up myriad new avenues for future investigation.
45. Dogovski C, Xie SC, Burgio G, et al. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance
. PLoS Biol 2015; 13:e1002132.
46. Hott A, Casandra D, Sparks KN, et al. Artemisinin
-resistant Plasmodium falciparum parasites exhibit altered patterns of development in infected erythrocytes. Antimicrob Agents Chemother 2015; 59:3156–3167.
47▪▪. Mbengue A, Bhattacharjee S, Pandharkar T, et al. A molecular mechanism of artemisinin resistance
in Plasmodium falciparum malaria
. Nature 2015; 520:683–687.
This study reports a novel candidate mechanism of artemisinin action and resistance. Artemisinins are proposed to target P. falciparum PI3K, leading to decreased PI3K activity, decreased PI3P levels, and inhibition of parasite growth. To overcome this effect, artemisinin-resistant parasites have evolved mutant K13-propeller proteins that fail to bind PI3K and to mark it for degradation. The resultant increases in PI3K activity and PI3P levels enable artemisinin-exposed parasites to continue their intraerythrocytic development until drug levels decrease and PI3K activity is restored. Future studies are needed to reconcile these findings with those of a recent population transcriptomics study showing that the PI3K transcript levels of artemisinin-sensitive and artemisinin-resistant parasites are not significantly different, and the widely-held belief that artemisinins act through an oxidation mechanism.
48. Leang R, Barrette A, Bouth DM, et al. Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010. Antimicrob Agents Chemother 2013; 57:818–826.
49. Chaorattanakawee S, Saunders DL, Sea D, et al. Ex vivo drug susceptibility and molecular profiling of clinical Plasmodium falciparum isolates from Cambodia in 2008–2013 suggest emerging piperaquine resistance
. Antimicrob Agents Chemother 2015; 59:4631–4643.
50. Lim P, Dek D, Try V, et al. Decreasing pfmdr1 copy number suggests that Plasmodium falciparum in Western Cambodia is regaining in vitro susceptibility to mefloquine. Antimicrob Agents Chemother 2015; 59:2934–2937.