Accepted Manuscript Title: How genomics is contributing to the fight against artemisinin-resistant malaria parasites Author: Pedro Cravo Hamilton Napolitano Richard Culleton PII: DOI: Reference:

S0001-706X(15)00103-5 http://dx.doi.org/doi:10.1016/j.actatropica.2015.04.007 ACTROP 3589

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

12-12-2014 6-4-2015 11-4-2015

Please cite this article as: Cravo, P., Napolitano, H., Culleton, R.,How genomics is contributing to the fight against artemisinin-resistant malaria parasites, Acta Tropica (2015), http://dx.doi.org/10.1016/j.actatropica.2015.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

How genomics is contributing to the fight against artemisininresistant malaria parasites Pedro Cravo1,2,3, Hamilton Napolitano2,4 and Richard Culleton5 1

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Laboratory of Genomics and Biotechnology (GenoBio), IPTSP, Universidade Federal de Goiás, Goiânia- GO, 74605050, Brazil 2

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PPSTMA, Centro Universitário de Anápolis (UniEVANGÉLICA), Anápolis-GO, 75083515, Brazil 3

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Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, 1349-008, Portugal 4

Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás. 459, 75001970, Anápolis, GO, Brazil Malaria Unit, Department of Pathology, Institute of Tropical Medicine, Nagasaki

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University, Nagasaki 8528523 Japan

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Correspondence should be addressed to Pedro Cravo; [email protected]

Abstract

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Plasmodium falciparum, the malignant malaria parasite, has developed resistance to

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artemisinin, the most important and widely used antimalarial drug at present. Currently confined to Southeast Asia, the spread of resistant parasites to Africa would constitute a

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public health catastrophe. In this review we highlight the recent contributions of genomics to our understanding how the parasite develops resistance to artemisinin and its derivatives, and how resistant parasites may be monitored and tracked in real-time, using molecular approaches.

1. Introduction

Plasmodium falciparum, the most pathogenic of the malaria parasites that infect man, kills around 600,000 people every year, mostly children, and mostly in Africa [1]. Control of this parasite is currently based on anti-mosquito measures, including the use of bednets (untreated or treated with insecticides) and case management through treatment of infected patients with antimalarial drugs. The long-term viability of effective treatment is jeopardized by the parasite’s intrinsic ability to evolve resistance to drugs [2]. Drug resistant mutant Plasmodia are often selected due to the parasite’s rapid genome replication rate [3], its relatively high mutation rate per generation [4], and the enormous numbers of parasites in existence at any given time [5]. The probability of the emergence

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of de novo mutants resistant to any given antimalarial drug is a function of the complexities of the mutation(s) required to confer drug tolerance and, ultimately, resistance to parasites. The speed with which such mutants are selected within populations depends primarily on the pharmacokinetics of the drug itself, and on the degree of usage within a population. Following the establishment of a pool of tolerant parasites to other locations where the drug is being used.

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and/or resistant parasites within a population, there often follows the spread of these

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Although sub-Saharan Africa countries bear the brunt of the malaria burden, a significant number of cases also occur in Southeast Asia and South America. Interestingly, despite

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the fact that more than 90% of the total global P. falciparum parasite biomass is found in Africa, drug resistance has historically emerged elsewhere, particularly South-east Asia, in so-called “resistance epicenters”, before spreading globally. Such epicenters are

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found in both South America and Southeast Asia, with resistance against numerous antimalarial drugs including chloroquine, pyrimethamine and sulphadoxine known to have emerged independently in both these regions [6-10]. Quite why it is these particular

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regions in which parasite drug resistance initially emerges is incompletely understood, but it is likely to be dependent on particular local demographical and epidemiological scenarios and the genetic constitution of the resident parasites. Due, perhaps, to human

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population movement demographics, and the inherent properties of the parasites on multiple occasions [11].

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themselves, drug resistant mutants from Southeast Asia appear to have spread to Africa

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[6,7][8,9]Due to the spread of multi-drug resistant P. falciparum parasites, the majority of countries where malaria is endemic have adopted artemisinin derivatives (ARTs) as their first-line therapies. ARTs have a short half-life but act extremely quickly in reducing parasite densities and symptoms [12]. ARTs are combined with chemically distinct drugs (such as mefloquine, amodiaquine, piperaquine, pyrimethamine/sulphadoxine, lumefantrine, pyronaridine or naphthoquine) in the form of artemisinin combination therapies (ACTs) for more favorable pharmacokinetics and to reduce the probability of mutations that underlie resistance and treatment failure emerging in parasite populations [13], since treatment is unlikely to encounter mutants that are resistant to both artemisinin derivatives and their partner drugs. Unfortunately, however, the efficacy of artemisinin is waning in specific areas of Southeast Asia, threatening millions of lives, because there are no effective alternative treatments available. The first signs of resistance to artemisinin derivatives (ARTs) in P. falciparum were observed along the Thai-Cambodian border [14,15], characterized by a

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delayed Parasite Clearance Time (PCT) in ACT-treated patients. Subsequently, delayed PCTs were reported from a neighboring area of the Mekong region [16,17]]. Additionally, due to the historical evidence surrounding drug resistance in malaria, there is now a wellfounded fear that resistance to ARTs may also arise independently in South America [20]. The risk that ART resistance may spread from the Mekong region to the rest of Asia

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and possibly to Africa or the prospect of resistance emerging independently in those

regions and in South America, has incited a broad agreement that developing

mechanisms to halt the spread of resistant parasites and preventing the establishment of

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new foci of resistance is now crucial. In line with this urgent priority, the World Health Organization orchestrated an official Global Plan for Artemisinin Containment (GPARC)

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that “sets out a high-level plan of attack to protect ACTs as an effective treatment for Plasmodium falciparum malaria” [21]. One of the main priorities of the GPARC is to

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increase monitoring and surveillance of ART resistance through a variety of different tools, towards providing evidence-based information that can rapidly inform actions to deter resistance.

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Identifying the genetic mutations underlying ART resistance and subsequently tracing them in parasite populations would offer an extremely powerful tool for resistance surveillance, as molecular markers can be used both in real-time or retrospectively to

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map the geographic origins and migration patterns of drug resistance [18]. This would

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allow the delimitation of areas of de novo resistance selection or of resistant parasite migration, and control programmes informed and adjusted appropriately.

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Understanding the mode of action of ARTs is the first step in elucidating how the parasite evolves resistance to these drugs. It is also important for identifying molecular markers to monitor resistance. Different molecular models have been proposed to explain ARTs’ mechanism of action against malaria parasites (reviewed in detail in Ding et al., 2011 [19]). Although the precise mechanism of action proposed by these different models is still under debate, there is a widely accepted view that ARTs are first activated through cleavage of the intrinsic peroxide bond after reacting with haem and ferrous ions, resulting in generation of free radicals that kill the parasite upon interacting with susceptible groups of several potential target proteins [20]. One of the first proteins to be suggested as a target of ARTs was the SERCA-type ATPase protein of P. falciparum (PfATPase6). Early studies suggested that resistance to ARTs could depend on mutation-driven alterations in PfATPase6 [21,22] or amplification of the “classic” multi-drug resistance gene 1, Pfmdr1 [23-26]. However, no correlation was found between these genes and in vivo resistance to artemisinins in the first

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confirmed cases of resistance along the Thai/Cambodian border [14,27-30]. Thus, until very recently validated genetic markers to monitor the evolution of artemisinin resistance in natural parasite populations were lacking. With the recent advances in genomics and the development of Whole Genome

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Sequencing (WGS) it has become possible to develop a new unbiased paradigm to investigate the genetic architecture of ART resistance, through whole genome typingphenotype association studies.

In this review we will focus on how these genomics

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approaches are providing unprecedented opportunities to accelerate both the discovery of genetic mutations linked to resistance to ARTs and the means by which they are being

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used for the real-time molecular surveillance of resistance.

resistance in the post-genomic era.

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2. Discovery and application of molecular markers for surveillance of drug

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In the pre-genomic era, the processes of identifying and applying phenotype-traceable mutations were slow and laborious because the tools of genetic analyses were essentially “low-throughput”. The publication of the P. falciparum genome in 2002 [31]

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heralded a new age in the investigation of the parasite’s biology, the so-called “post-

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genomic” era. The knowledge of the complete sequence of the P. falciparum genome, added to previous studies on conservation of gene order between different species (gene synteny) [24], provided new ways of identifying genetic markers involved in the

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control of phenotypes such as drug resistance. Prior to this, advances in understanding the underlying genetics of drug resistance in malaria parasites relied, to a large extent, on the labour intensive production of genetic-crosses, their selection, and analyses based on a limited number of markers distinguishing different parasite strains [3]. It was through the use of genetic crosses that the discovery was made of mutations in the pfdhfr and pfcrt genes, implicated in pyrimethamine and chloroquine resistance, respectively [25,26]. However, these mutations were identified long after resistance to the drugs had emerged and spread throughout the world. Today, however, the availability of the full genome sequences of many species of malaria parasites and the more recent advent of high-throughput nucleic acid sequencing, or Whole Genome Sequencing (WGS) [27], have dramatically improved our ability to identify drug resistance mutations de novo, and have expanded the means by which they can be used to monitor resistance.

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One extremely efficient and informative method of identifying drug resistance-associated genes in malaria parasites is to apply drug pressure in order to select for resistance in the laboratory with P. falciparum (in vitro) or in experimental animal models of malaria (in vivo) (Figure 1). The strength of the experimental selection approach is that the genome of the resulting drug-resistant parasite should be identical to that of its sensitive

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progenitor, save for the mutations arising during the evolution of resistance. By using

WGS, it is then possible to very rapidly obtain the whole genome sequence of the drugresistant parasite and to compare it with that of its sensitive progenitor [28]. This allows

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the unbiased identification of all the genetic variation between the two parasites, including those mutations that functionally determine the drug resistance phenotype,

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mutations that may be compensatory, and random mutations acquired due to prolonged in vitro culture or in vivo passage. The role of these newly discovered mutations for the evolution of resistant phenotypes in natural parasite populations from malaria-endemic

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areas can then be assessed by population genetics approaches and/or Genome Wide Association Studies (GWAS).

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Population genetics is a potent tool for mutation discovery and for monitoring drug resistance in the field, since the emergence and spread of antimalarial resistance often leaves a genetic signature that can be detected and characterized. Such signatures,

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known as “selective sweeps” are characterized by a marked reduction of genetic

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diversity around the genomic locus involved in resistance to a given drug, and have been well defined for the pfcrt, dhps and dhfr genes, determining resistance to chloroquine,

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sulphadoxine and pyrimethamine, respectively [11]. Signatures of selective sweeps in parasite populations containing drug resistant mutants have been identified through population genetics approaches [32-35]. Additionally, specific patterns of selective sweeps might hold important clues about the strength of selection and the evolutionary history of the resistance mutations. The ability to rapidly obtain the full genome sequence of multiple parasite isolates is allowing a paradigm shift from “population genetics” to “population genomics”, significantly improving our analytic resolution which was traditionally reliant on a limited number of genetic markers. Likewise, genome-wide association studies (GWAS) for both the identification of new drug resistance genes and for resistance monitoring benefit tremendously from the genotyping accuracy delivered by post-genomics advances in high throughput nucleic acid sequencing. GWAS compare the DNA of drug-sensitive and resistant parasites from natural populations by inspecting the genome-wide variation between both to check if any variant is associated with resistance to a particular drug. If one type of the variant (allele) is more frequent in drug-resistant parasites, the genetic

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mutation (often an SNP) is assumed to be associated with resistance. Provided enough genetic variants are analyzed, the associated SNPs are then used to mark a region of the parasite’s genome where the mutated gene is located. Because GWAS consider the entire genome, it is unbiased and non-candidate-driven in contrast to approaches that specifically examine one or a few genetic regions. GWAS have already proved

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successful in the de novo identification of a gene involved in increased resistance to

halofantrine [36] and have recently been validated as a powerful tool for identifying

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several other malaria drug resistance-associated loci [37].

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3. The chromosome XIII locus of P. falciparum and artemisinin resistance surveillance

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The recent discovery of a locus on chromosome XIII associated with delayed PCT following treatment of clinical malaria with ART in South-east Asia has profoundly improved our capacity to monitor ART-resistant malaria. To this purpose, Cheeseman et

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al (2012) [35] used a used a two-phase strategy to identify regions of the P. falciparum genome that may be under ART selection in the area of suspected resistance in South-

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east Asia. In order to detect recent selective sweeps by ARTs, they first assessed geographical differentiation and haplotype structure at nearly 7000 SNPs in 91 parasite

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samples from Laos, Cambodia and Thailand, that present low levels of genetic differentiation between them, but differences in PCTs after ART treatment. Thirty-three

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regions of the genome with evidence of being under selection were thus identified. Further screening of these regions using SNP and microsatellite typing in clinical samples from Thailand with well documented PCTs after ART treatment, provided evidence of a recent selective sweep in a ≈ 35Kb region of chromosome XIII that was strongly associated with delayed PCTs [35]. Subsequently, Takala-Harrison et al (2013) [38] carried out a GWAS study using phenotypic data from four clinical trials evaluating the effect of artesunate monotherapy in Western Cambodia, where resistance to ART derivatives was suspected. They then collected parasite samples from the participants in the trials and genotyped 8000+ SNPs using a P. falciparum specific SNP array. In order to identify regions of the genome linked to artesunate resistance, parasite genomes were inspected for signs of recent positive selection and the PCT of each sample was correlated to its genotype. The resulting analysis identified four ART resistanceassociated SNPs located on chromosomes I (one), XIII (two) and XIV (one). Interestingly, the two SNPs on chromosome XIII were located in a region of the genome that was suggested to be under recent strong positive selection in Cambodia. A follow-up

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genetic fingerprinting study not only confirmed the finding that the region on chromosome XIII is indeed a signature of ART resistance in South-east Asia [39], but also elucidated the pattern of parasite population structure at the epicentre of artemisinin resistance in western Cambodia. By surveying the genomes of around 800 parasite strains originating from different geographical settings across Africa and Southeast Asia,

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including the emerging local resistant parasites, the study revealed several artemisinin-

resistant strains of the parasite that appear to be spreading relatively quickly through the

local parasite population in Western Cambodia [39]. Interestingly, the genetic profile and

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population structure of the parasites from Western Cambodia was extremely different to those from other regions, including not just the samples from Africa, but also those from

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neighboring countries. One important feature of these genetic analyses is that it allows the rapid identification of resistant parasites even if the genetic mutations responsible for resistance are unknown. Therefore, this approach represents an extremely informative

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tool for global molecular surveillance in order to trace the origins and geographical trajectories of artemisinin-resistant parasites in real time.

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Although the studies described above clearly demonstrated the correlation between a locus on chromosome XIII of P. falciparum and ART PCTs, no specific mutations could be unequivocally linked to the phenotype. A subsequent study that selected an ART-

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resistant parasite in vitro, then identified mutations in a putative Kelch gene located

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within the locus, through WGS comparison of the mutant genome with that of its

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sensitive progenitor (see section 4 below).

4. Artificial in vitro selection and GWAS identify the K-13 propeller gene as a molecular marker of ART resistance. Artificial selection of ART resistant mutants in vitro combined with WGS for mutation detection has been used recently in P. falciparum to identify a novel molecular marker of ART resistance. Ariey et al (2014) [40] selected an ART-resistant parasite line, denoted F32-ART5, by culturing an ART-sensitive clone under 125-cycles of escalating doses of ART for five years. They then sequenced the whole genomes of the F32-ART5 resistant parasites and a sibling line, F32-TEM, which was cultured in parallel without artemisinin. They identified eight point mutations distributed across seven genes in the F32-ART5 line compared to ART-sensitive parasites. One of these genes, a cysteine protease falcipain 2a (PF3D7_1115700), had been previously associated with in vitro ART resistance [41], but the others were new. Among the genes identified de novo in that

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study, mutations in a particular gene located in a region of chromosome XIII previously associated with ART clearance phenotypes [38], encoded a putative Kelch protein (PF3D7_1343700). These mutations directly correlated with increased in vitro responses to ART among 49 culture adapted parasite isolates assayed using the ring-stage survival assay (RSA), previously shown to reflect Clinical ART resistance [39]. To further explore

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the role of the Kelch gene (hereafter termed the Pf-K13 propeller) in ART resistance in

natural parasite populations, the authors analyzed a set of parasite samples archived between 2001 and 2012 from drug trials in Cambodia, and confirmed that mutations in

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the gene were associated with the slow clearance phenotype. Subsequently, using as

GWAS approach, Takala-Harrison et al (2014) showed that, as feared, resistant

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parasites carrying mutant Pf-K13 propeller alleles from Cambodia have already migrated to Thailand and Vietnam [40]. Importantly, they also discovered two regions in Myanmar where mutations in the Pf-K13 propeller were not imported from neighboring locations,

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but rather appeared to have evolved independently. Subsequently, a large multicenter GWAS not only confirmed the above findings, but went further to elucidate the genetic

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architecture of ART-resistant P. falciparum, using data from over a thousand samples distributed across 15 different locations in Southeast Asia [42]. Several mutations in the Pf-K13 propeller domains where associated with delayed PCTs following treatment with

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ARTs, confirming the results of previous studies. Interestingly, a number of nonsynonymous polymorphisms in other genes on different chromosomes (Table 1), such as

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fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) were also significantly associated

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with slow PCTs ([42], Table 1). Analysis of the parasite population structure indicated that the above mutations are markers of a common genetic background in Southeast Asia, on which Pf-K13 mutations are more prone to evolve, suggesting that the probability of new Pf-K13 mutations arising increases under certain predisposing genetic features [42]. As such, these polymorphisms can also be applied as early warning sentinels to predict future evolution of resistance to ARTs in natural populations of P. falciparum.

In order to investigate the molecular epidemiology of putative Pf-K13 propeller artemisinin resistance genotypes in African parasite populations Taylor et al recently developed and applied a pooled deep-sequencing approach assay to quantify rare polymorphisms in over a 1000 parasite samples collected from 2002 from various locations across sub-Saharan Africa [43]. They did not detect the SNPs that are associated with ART resistance in Southeast Asian parasites. However, they found a significant degree of diversity in the gene (15 coding mutations) consistent with a large

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pool of naturally occurring genetic variation. Since no signs of a selective sweep in or around the Pf-K13 propeller gene were detected, the effect of the resident mutants on artemisinin sensitivity is unknown [43]. Nevertheless, the study has provided a contemporary snapshot of Pf-K13 propeller genetic constitution that will complement both ongoing and future studies on the molecular surveillance of ART resistance in sub-

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Saharan Africa.

5. Additional candidate markers for ART resistance surveillance

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discovered through a rodent malaria model

The rodent malaria parasite Plasmodium chabaudi [44] provides a fast track tool for the

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rapid identification of candidate genes controlling resistance to ARTs, through a combination of experimental evolution, classical genetics and WGS. This experimental system has identified genetic mutations in parasites artificially selected in vivo for

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resistance to artemisinin (ART) and artesunate (ATN). These mutations encode alterations in a de-ubiquitinating enzyme (PcUBP-1) [45] involved in the ubiquitinproteasome pathway and in a gene encoding a putative adaptor protein subunit (Pcap2-

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mu) [44], thought to mediate the traffic of haemoglobin (Hb) into the parasite food vacuole where Hb is digested [46]. Since the detrimental role of ART on both the

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ubiquitin-proteasome pathway and in Hb degradation has been proposed or demonstrated [45,47], there is a well-founded premise for the involvement of the ubp-1

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and ap2-mu genes in resistance to ARTs. The real value of mutations in these genes as markers to monitor ART resistance in human malaria parasites rests on the evaluation of their association with ART phenotypes in natural populations of P. falciparum. An independent study that involved the analysis of SNPs from WGS data in 27 Kenyan isolates of P. falciparum whose in vitro response to several drugs was assessed, identified genetic variants associated with susceptibility to dihydroartemisinin that implicated a region on chromosome XIII, as well as the Pfubp1 gene, the orthologue of the Pcubp1 gene [48], located on chromosome I,. Additionally, they also detected a strong signal of recent positive selection surrounding the Pfubp1 locus, suggesting that the gene may be under selection by ART. A potential role for Pfubp1 as well as Pfap2mu (whose orthologues were originally identified in the rodent model) in the modulation of ART treatment outcome in vivo was recently emphasized in the work of Henriques et al (2014) [49] who detected directional selection at both loci in P. falciparum-infected Kenyan children treated with ACT.

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6. Conclusion Artemisinin-based Combination Therapies (ACTs) have been the mainstay of treatmentbased malaria control in recent years, contributing to a significant reduction in the number of cases and deaths worldwide [1]. However, the recent evolution of resistance

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to artemisinin derivatives in P. falciparum parasites from Southeast Asia may seriously threaten the progress achieved thus far because there are no alternative effective treatments available and no efficacious malaria vaccines in sight. Elimination of P.

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falciparum in this region would be the ideal solution to prevent the spread of ART resistance to other malaria endemic regions. Meanwhile, the priorities are now to prevent

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ART resistance from spreading to Africa and/or to the neighboring countries, such as Vietnam, India and China and to monitor possible sites where it may evolve independently, particularly in South America, where demographic and epidemiological

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scenarios favor the emergence of resistance. The recent discovery, validation and application of the Pf-K13 propeller gene as a molecular marker for surveillance of artemisinin resistance in South-East Asia is allowing mapping and tracking the paths of

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resistant parasites in that recently identified epicenter of resistance. It remains to be established whether the Pf-K13 propeller will represent a “universal” marker for surveillance of ART resistance worldwide [49]. Nevertheless, this and other markers

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discovered through genomics approaches can now be further evaluated and used in

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Conflict of Interests

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real-time for molecular surveillance of artemisinin resistance.

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgements

Pedro Cravo and Hamilton Napolitano are productivity fellows of the National Council of Technological and Scientific Development (CNPq) of Brazil.

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Candidate marker(s)

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TABLE 1: Main candidate molecular markers currently used or under investigation for surveillance of artemisinin resistance in Plasmodium falciparum (* The major determinant of resistance in Southeast Asian parasites) Chromosome

Type of study and evidence

XIII

GWAS. 35Kb locus where several SNPs were associated with ART PCTs and suggested to be under strong recent positive selection

DNA repair protein RAD5, putative (RAD5) (PF3D7_1343400)

XIII

GWAS. Associated with ART PCTs. Two SNPs (one of them 690 bp downstream PF3D7_1343400) are in a region of chromosome 13 that appears to have been under strong recent positive selection.

ABC transporter, (heavy metal transporter family), putative (PF3D7_1352100)

XIII

Kelch protein K13*(PF3D7_1343700)

XIII

(PF3D7_1318100)

XIII

[35]

[36]

GWAS. Previously associated with drug resistance

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Ferredoxin, putative (fd)

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Several genes in a 35Kb region of chromosome 13

Reference(s)

Experimental evolution combined with WGS and GWAS. SNPs were associated with increased resistance in an P. falciparum artemisininresistant parasite line selected in the laboratory and with delayed parasite clearance in clinical isolates from Cambodia and from several other locations across Southeast Asia.

[37]

[38,40, 42]

[42]

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XIV

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Apicoplast ribosomal protein S10 precursor, putative (arps10)

Multidrug resistance protein 2+ (heavy metal transport family) (MDR2)

XIV

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(PF3D7_140900.1)

GWAS. SNPs were associated with delayed parasite clearance in clinical isolates from several locations across Southeast Asia.

(PF3D7_1447900) VII

(PF3D7_0709000)

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Ubiquitin carboxyl-terminal hydrolase 1, putative (UBP1) (PF3D7_0104300)

XI

Experimental evolution combined with WGS and gene KO studies. SNPs were associated with increased resistance in an P. falciparum artemisininresistant parasite line selected in the laboratory and KO mutants have significantly decreased ART sensitivity

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Cysteine proteinase falcipain 2a (FP2A) (PF3D7_1115700)

Adaptor protein subunit, putative (AP2-mu) (PF3D7_1218300)

I

XII

[42]

[42]

ed

Chloroquine resistance transporter (CRT)

[42]

Experimental evolution combined with WGS in the rodent malaria model P. chabaudi. Associated with in vitro and ART PCTs in samples from Kenya and suggested to be under strong recent positive selection. Experimental evolution combined with WGS in the rodent malaria model P. chabaudi. Associated with ART PCTs in samples from Kenya.

[38,50]

[28,47,48] [44,48]

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FIGURE 1: Experimental evolution combined with Whole Genome Sequence for identifying molecular markers of drug resistance in malaria parasites

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Legend: Drug sensitive parasites are grown in vivo (A) or in vitro (B) under escalating drug concentrations for many generations until a drug-resistant parasite is selected. The genome of the drug-resistant parasite will differ from that of its sensitive progenitor at a limited number of sites, some of which will be directly related to the resistance phenotype. The two genomes can be rapidly re-sequenced through Whole Genome Sequencing (WGS) and compared and the genes harboring candidate mutations are identified. The contribution of each of the mutations for the expression of the drug resistance phenotype can be evaluated though genetic crossing, genetic transfection experiments and/or association studies in natural parasite populations. If validated, they can then be used as markers for molecular surveillance of drug resistance. Candidate molecular markers of ART resistance have been successfully identified through this approach recently [28,38,44].

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Highlights

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Plasmodium falciparum has developed resistance to artemisinin. The genetic basis for this resistance is now becoming clear We discuss how genomics approaches can reveal how resistance arises These approaches can also be used to develop markers to monitor drug resistance

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Graphical Abstract (for review)

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How genomics is contributing to the fight against artemisinin-resistant malaria parasites.

Plasmodium falciparum, the malignant malaria parasite, has developed resistance to artemisinin, the most important and widely used antimalarial drug a...
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