Ex Vivo Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Western,Northern, and Eastern Cambodia, 2011-2012: Association with Molecular Markers

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC₅₀s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC₅₀s (GMIC₅₀s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC₅₀s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC₅₀ and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.     

Ex Vivo Activity of Endoperoxide Antimalarials,Including Artemisone and Arterolane,against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC₅₀s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC₅₀ susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC₅₀s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.     

pfmdr1 Amplification and Fixation of pfcrt Chloroquine Resistance Alleles in Plasmodium falciparum in Venezuela

Molecular tools are valuable for determining evolutionary history and the prevalence of drug-resistant malaria parasites. These tools have helped to predict decreased sensitivity to antimalarials and fixation of multidrug resistance genotypes in some regions. In order to assess how historical drug policies impacted Plasmodium falciparum in Venezuela, we examined molecular changes in genes associated with drug resistance. We examined pfmdr1 and pfcrt in samples from Sifontes, Venezuela, and integrated our findings with earlier work describing dhfr and dhps in these samples. We characterized pfmdr1 genotypes and copy number variation, pfcrt genotypes, and proximal microsatellites in 93 samples originating from surveillance from 2003 to 2004. Multicopy pfmdr1 was found in 12% of the samples. Two pfmdr1 alleles, Y184F/N1042D/D1246Y (37%) and Y184F/S1034C/N1042D/D1246Y (63%), were found. These alleles share ancestry, and no evidence of strong selective pressure on mutations was found. pfcrt chloroquine resistance alleles are fixed with two alleles: S_tctVMNT (91%) and S_agtVMNT (9%). These alleles are associated with strong selection. There was also an association between pfcrt, pfmdr1, dhfr, and dhps genotypes/haplotypes. Duplication of pfmdr1 suggests a potential shift in mefloquine sensitivity in this region, which warrants further study. A bottleneck occurred in P. falciparum in Sifontes, Venezuela, and multidrug resistance genotypes are present. This population could be targeted for malaria elimination programs to prevent the possible spread of multidrug-resistant parasites.Amplification of the Plasmodium falciparum multidrug resistance gene (pfmdr1) has been implicated in mefloquine (MQ) resistance in Thailand and Cambodia (1, 17, 27, 28, 34, 41), but not elsewhere. It is not known if amplification has occurred in Venezuela, where MQ monotherapy was used between 2001 and 2004 and the combination of artesunate (AS) and MQ thereafter. pfmdr1 duplication is also implicated in resistance to lumefantrine, halofantrine, quinine, and AS (39) and may decrease resistance to chloroquine (CQ) (43). Also, single-nucleotide mutations in pfmdr1, such as N86Y, Y184F, S1034C, N1042D, and D1246Y (the mutated amino acid is shown in boldface type), are postulated to modulate drug response. While these mutations may or may not contribute to CQ resistance (40), mutations at codons 1034, 1042, and 1246 make parasites more sensitive to MQ (40). Studies suggest at least two lineages of mutant pfmdr1 genotypes have evolved in South America (4, 21).In South America, CQ and sulfadoxine-pyrimethamine (SP) were used to treat P. falciparum prior to the use of artemisinin-based combination therapy (ACT). Resistance to CQ and SP evolved independently in South America (18, 23). Point mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene are correlated with CQ resistance (10). The pfcrt point mutation K76T is critical, but C72S, M74I, N75E, and N75K are also associated with resistance (48). There are at least four different origins of CQ resistance pfcrt alleles: one in Papua New Guinea (SVMNT), where the genotype represents amino acids at codons 72 to 76, one in Southeast Asia (CVIET) that spread to Africa, and two in South America (SVMNT/CVMNT in Brazil/Peru and CVMET/CVMNT in Ecuador/Colombia) (49).Molecular surveillance showed that, after drug removal, CQ resistance genotypes, in Malawi and China (16, 46), and SP resistance genotypes, in the Peruvian Amazon (52), declined. Therefore, the reduction in the frequency of resistant parasites likely occurred because resistant parasite populations are at a fitness disadvantage in the absence of drug pressure. In Bolivar State, Venezuela, mutant pfcrt alleles remained after the removal of CQ in 1986 (6) and mutant dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes remained fixed after SP removal (19). Whether the recent use of MQ and AS-MQ led to the evolution of pfmdr1 genotypes associated with AS and MQ resistance is unknown.This study in the state of Bolivar, Venezuela assessed the following: (i) whether pfmdr1 duplication has occurred, (ii) the frequency of pfmdr1 and pfcrt mutations, (iii) whether MQ and CQ drug pressure has affected variation surrounding these genes, and (iv) linkage disequilibrium between dhfr, dhps, pfcrt, and pfmdr1 alleles.(Part of this research [some data pertaining to pfmdr1 and pfcrt genotypes, microsatellites, and copy number] was presented at the 57th Annual Meeting of the American Society of Tropical Medicine and Hygiene, New Orleans, LA, 2008, and the 58th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Washington, DC, 2009.)     

Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

Cambodia''s first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC₅₀] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.     

Exploring the Contribution of Candidate Genes to Artemisinin Resistance in Plasmodium falciparum

The reduced in vivo sensitivity of Plasmodium falciparum has recently been confirmed in western Cambodia. Identifying molecular markers for artemisinin resistance is essential for monitoring the spread of the resistant phenotype and identifying the mechanisms of resistance. Four candidate genes, including the P. falciparum mdr1 (pfmdr1) gene, the P. falciparum ATPase6 (pfATPase6) gene, the 6-kb mitochondrial genome, and ubp-1, encoding a deubiquitinating enzyme, of artemisinin-resistant P. falciparum strains from western Cambodia were examined and compared to those of sensitive strains from northwestern Thailand, where the artemisinins are still very effective. The artemisinin-resistant phenotype did not correlate with pfmdr1 amplification or mutations (full-length sequencing), mutations in pfATPase6 (full-length sequencing) or the 6-kb mitochondrial genome (full-length sequencing), or ubp-1 mutations at positions 739 and 770. The P. falciparum CRT K76T mutation was present in all isolates from both study sites. The pfmdr1 copy numbers in western Cambodia were significantly lower in parasite samples obtained in 2007 than in those obtained in 2005, coinciding with a local change in drug policy replacing artesunate-mefloquine with dihydroartemisinin-piperaquine. Artemisinin resistance in western Cambodia is not linked to candidate genes, as was suggested by earlier studies.Antimalarial drug resistance is the single most important threat to global malaria control. Over the past 40 years, as first-line treatments (chloroquine and sulfadoxine-pyrimethamine) failed, the malaria-attributable mortality rate rose, contributing to a resurgence of malaria in tropical countries (11). In the last decade, artemisinins, deployed as artemisinin combination therapies (ACTs), have become the cornerstone of the treatment of uncomplicated falciparum malaria (20) and, in conjunction with other control measures, have contributed to a remarkable decrease in malaria morbidity and mortality in many African and Asian countries (4). The recent confirmation of the reduced artemisinin sensitivity of Plasmodium falciparum parasites in western Cambodia has therefore alarmed the malaria community (6). A large containment effort has been launched by the World Health Organization, in collaboration with the national malaria control programs of Cambodia and neighboring Thailand. The resistant phenotype has not been well characterized and is not well reflected by the results of conventional in vitro drug susceptibility assays. No molecular marker has been identified, which impedes surveillance studies to monitor the spread of the resistant phenotype. Identification of molecular markers would give insight into the mechanisms underlying artemisinin resistance and the mechanism of antimalarial action of the artemisinins.Mutations in several candidate genes have been postulated to confer artemisinin resistance. (i) P. falciparum mdr1 (pfmdr1) encodes the P-glycoprotein homologue 1 (Pgh1), which belongs to the ATP-binding cassette transporter superfamily, members of which couple ATP hydrolysis to the translocation of a diverse range of drugs and other solutes across the food vacuole and plasma membranes of the parasite (Fig. ​(Fig.1)1) (5). The gene is located on chromosome 7, is 4.2 kb in length, and contains only one exon. Mutations in and, more importantly, amplification of the wild-type gene confer resistance to the 4-methanolquinoline mefloquine, presumably through an increased ability to efflux the drug (15, 16). Mutations and amplification of the gene have also been associated with reduced in vitro susceptibility to the artemisinins (7, 16). In vivo selection of the pfmdr1 86N allele after artemether-lumefantrine treatment has been observed in Africa (17).Open in a separate windowFIG. 1.Predicted structure and representative haplotypes of P. falciparum multidrug resistance transporter. PfMDR1 is predicted to have 12 transmembrane domains, with its N and C termini located on the cytoplasmic side of the digestive vacuole membrane (adapted from reference 19). Mutations identified in pfmdr1 full-length sequences from Pailin and WangPha are indicated by the red circles. aa, amino acid.(ii) P. falciparum ATPase6 (pfATPase6) encodes the calcium-dependent sarcoplasmic/endoplasmic reticulum calcium ATPase, which was shown to be a target for the artemisinin drugs in Xenopus oocytes (8). The gene is 4.3 kb in length and has three exons on chromosome 1. A single amino acid change in pfATPase6, L263E, is associated with resistance to artemisinins in this model (8, 18). Mutation S769N in pfATPase6 in P. falciparum isolates from French Guiana was associated with decreased in vitro sensitivity to artemether (10). However, it is unclear whether mutations in pfATPase6 are associated with artemisinin resistance in vivo (1).(iii) The electron transport chain in the mitochondrial inner membrane is key to the malaria parasite''s capacity to produce ATP. Since activation of the endoperoxide bridge in the artemisinins by an electron donor is central to their antimalarial activity, mitochondrial proteins are potential activation sites for the artemisinins. Mutations in the mitochondrial genome, which is 6 kb long and which contains three genes (cytochrome b, COXI, COXIII), could therefore potentially change susceptibility to the artemisinins.(iv) ubp-1, a 3.3-kb gene located on chromosome 2, encodes a deubiquitinating enzyme. Mutations V739F and V770F in ubp-1 of P. chabaudi were recently identified by linkage group analysis of an elegant genetic-cross experiment to confer resistance to artesunate in this rodent malaria parasite (9).(v) Laboratory-induced artemisinin resistance in the P. chabaudi model has been demonstrated in a chloroquine-resistant strain. This suggests that chloroquine resistance in this model might be a prerequisite for the subsequent development of artemisinin resistance. We therefore also assessed the parasite genome for the presence of the P. falciparum CRT (pfCRT) K76T mutation, which plays a central role in the chloroquine resistance of P. falciparum.We report here the molecular characteristics of these five groups of genes in P. falciparum isolates from western Cambodia, where most infections show reduced sensitivity to artesunate, compared to those of strains obtained from northwestern Thailand, where infections are artemisinin sensitive (6).     

Synthesis, antimalarial activity, and intracellular targets of MEFAS, a new hybrid compound derived from mefloquine and artesunate

A new synthetic antimalarial drug, a salt derived from two antimalarial molecules, mefloquine (MQ) and artesunate (AS), here named MEFAS, has been tested for its pharmacological activity. Combinations of AS plus MQ hydrochloride are currently being used in areas with drug-resistant Plasmodium falciparum parasites; although AS clears parasitemia in shorter time periods than any other antimalarial drug, it does not cure infected patients; in addition, MQ causes side effects and is rather expensive, important problems considering that malaria affects mostly populations in poor countries. Here, we show that MEFAS is more effective than the combination of AS and MQ, tested in parallel at different mass proportions, against P. falciparum (chloroquine-resistant clone W2 and chloroquine-sensitive clone 3D7) in vitro and in mice infected with Plasmodium berghei, promoting cure of this infection. MEFAS tested against HepG2 hepatoma cells exhibited lower toxicity than the antimalarials AS and MQ alone or combined. Possible targets of MEFAS have been studied by confocal microscopy using fluorescent probes (Fluo-4 AM and BCECF-AM) in P. falciparum synchronous culture of W2-infected red blood cells. Dynamic images show that MEFAS exhibited intracellular action increasing cytoplasmic Ca²⁺ at 1.0 ng/ml. This effect was also observed in the presence of tapsigargin, an inhibitor of SERCA, suggesting an intracellular target distinct from the endoplasmic reticulum. Trophozoites loaded with BCECF-AM, when treated with MEFAS, were still able to mobilize protons from the digestive vacuole (DV), altering the pH gradient. However, in the presence of bafilomycin A1, an inhibitor of the H⁺ pump from acidic compartments of eukaryotic cells, MEFAS had no action on the DV. In conclusion, the endoplasmic reticulum and DV are intracellular targets for MEFAS in Plasmodium sp., suggesting two modes of action of this new salt. Our data support MEFAS as a candidate for treating human malaria.     

Role of pfmdr1 Amplification and Expression in Induction of Resistance to Artemisinin Derivatives in Plasmodium falciparum

Artemisinin and its derivatives are the most rapidly acting and efficacious antimalarial drugs currently available. Although resistance to these drugs has not been documented, there is growing concern about the potential for resistance to develop. In this paper we report the selection of parasite resistance to artelinic acid (AL) and artemisinin (QHS) in vitro and the molecular changes that occurred during the selection. Exposure of three Plasmodium falciparum lines (W2, D6, and TM91C235) to AL resulted in decreases in parasite susceptibilities to AL and QHS, as well as to mefloquine, quinine, halofantrine, and lumefantrine. The changes in parasite susceptibility were accompanied by increases in the copy number, mRNA expression, and protein expression of the pfmdr1 gene in the resistant progenies of W2 and TM91C235 parasites but not in those of D6 parasites. No changes were detected in the coding sequences of the pfmdr1, pfcrt, pfatp6, pftctp, and pfubcth genes or in the expression levels of pfatp6 and pftctp. Our data demonstrate that P. falciparum lines have the capacity to develop resistance to artemisinin derivatives in vitro and that this resistance is achieved by multiple mechanisms, to include amplification and increased expression of pfmdr1, a mechanism that also confers resistance to mefloquine. This observation is of practical importance, because artemisinin drugs are often used in combination with mefloquine for the treatment of malaria.Plasmodium falciparum parasites have developed resistance to conventional antimalarial drugs by various means, including alteration of the enzymes targeted by drugs (8, 23, 24, 32) and mutation or amplification of the genes coding for proteins involved in drug transport (13, 34, 35). One of these proteins, P. falciparum multidrug resistance transporter 1 (PfMDR1), or Pgh1, a P. falciparum homologue of mammalian P-glycoprotein (15, 45), has been implicated in resistance to several structurally different antimalarial compounds. In early studies, exposure of P. falciparum laboratory lines to mefloquine resulted in amplification of the pfmdr1 gene (45), with concomitant increases in resistance to mefloquine (MQ), quinine (QN), and halofantrine (HF) (7, 30, 31, 45). Amplification of pfmdr1 was also observed in field isolates from different geographical locations (4, 34, 35, 42). Increased pfmdr1 copy numbers (CN) in field isolates were associated with higher inhibitory concentrations (IC) of MQ, QN, HF, and artemisinin (QHS) in vitro (34, 46) and were linked to the failure of MQ monotherapy and mefloquine-artesunate combination therapy in studies conducted in Thailand and on the Thai-Cambodian border (2, 35). Furthermore, direct evidence of the role of pfmdr1 in the modulation of parasite susceptibility came from a report where inactivation of 1 of 2 copies of pfmdr1 in the P. falciparum FCB line led to moderate increases in susceptibilities to artemisinin and arylaminoalcohol drugs (39).In addition to gene amplification, several polymorphic positions in the pfmdr1 gene (N86Y, Y184F, S1034C, N1042D, and D1246Y) have been identified in field isolates (14) and have been shown to contribute to altered parasite responses to QN, HF, MQ, chloroquine (CQ), and QHS in vitro (10, 28, 33, 36, 39). In particular, the last three mutations (S1034C, N1042D, and D1246Y) are implicated in increased sensitivity to artemisinin over that of the “wild type” (with S, N, and D) (36). Conversely, significant decreases in susceptibilities to QHS, MQ, and HF are observed when the “wild-type” N at position 1042 is restored (39). These findings are consistent with the early observation that the “wild-type” pfmdr1 allele (with N, Y, S, N, and D) is associated with reduced susceptibilities of the progeny of the genetic cross of the P. falciparum 3D7 and HB3 lines to MQ, HF, QHS, and artemether (10).Although PfMDR1 is clearly implicated in the modulation of parasite responses to antimalarial drugs, including artemisinins, the mechanism of its action is largely unknown. A recent study using heterologous expression of PfMDR1 in Xenopus laevis oocytes demonstrated that some drugs, including HF, QN, and CQ, are substrates for PfMDR1 (38). It is not clear whether artemisinins interact with PfMDR1. Several proteins have been shown to interact with artemisinin. The translationally controlled tumor protein (TCTP) binds to radioactively labeled dihydroartemisinin (5) and is overexpressed in rodent Plasmodium yoelii parasite lines with decreased susceptibility to artemisinin (44). Another protein that may interact with artemisinins is the sarcoplasmic reticulum Ca²⁺ ATPase 6 (PfATP6); this enzyme, when expressed in Xenopus oocytes, was specifically inhibited by artemisinin derivatives containing an endoperoxide bridge (11). In addition, the activity of the enzyme was greatly influenced by the introduction of several mutations (e.g., L263E) (43). Furthermore, analysis of naturally occurring polymorphisms in PfATP6 in field isolates from French Guiana suggested that a polymorphism at codon 769 may be associated with reduced susceptibility of these isolates to artemether in vitro (19). However, subsequent reports failed to detect codon 263 or 769 polymorphisms in the field (12, 27, 48).Although resistance to artemisinins has not been documented in the field, induction of artemisinin resistance in vitro may help in the identification of molecular markers and drug target sites as well as in designing strategies for combating artemisinin resistance when it arises.Several attempts have been made to develop resistance to artemisinin derivatives in P. falciparum (18, 20, 47) in vitro. Inselburg (18) induced resistance to artemisinin by using mutagens, but these lines are no longer available for study. Other attempts to select resistance with increasing drug pressure have led to various endpoints. Jiang (20) produced a 3-fold decrease in susceptibility to sodium artesunate (AS), but resistance proved unstable. Yang et al. (47) achieved 8.9-fold resistance to AS, although few data are available about the stability of the resistance selected or the methods used. A recent study reported the development of stable resistance to QHS and AS in the rodent parasite Plasmodium chabaudi chabaudi (1). Linkage group analysis of the resistant progeny from the same parental parasites identified two nonsynonymous mutations occurring independently in the P. chabaudi putative ubiquitin carboxyl-terminal hydrolase gene (pcubp1, or pcubcth): V739F appeared after selection with artesunate, whereas V770F occurred in progeny selected with CQ (17). No new mutations in PcUBP1 were detected after further selection with QHS. Attempts to develop stable resistance in the P. falciparum NF54 and 7G8 parasite lines were unsuccessful; parasites reverted to the sensitive phenotype after cryopreservation (17).Here we report the selection of resistance to artelinic acid (AL) and to QHS and its derivatives in vitro in several P. falciparum lines of different genetic backgrounds. We also investigated the possible mechanisms involved in the development of resistance. We present evidence that pfmdr1 gene amplification and expression are required in order for some, but not all, parasites to withstand high concentrations of AL or QHS in vitro.     

Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment

Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia (P <10⁻³). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC₅₀) of PP were independent of treatment outcomes, in contrast to median mefloquine IC₅₀, which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P = 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P = 0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.)     

Role of Pfmdr1 in In Vitro Plasmodium falciparum Susceptibility to Chloroquine,Quinine, Monodesethylamodiaquine,Mefloquine, Lumefantrine,and Dihydroartemisinin

The involvement of Pfmdr1 (Plasmodium falciparum multidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (P = 0.0023), LMF (P = 0.0001), DHA (P = 0.0387), and MQ (P = 0.00002). The N86Y mutation was not associated with CQ (P = 0.214) or QN (P = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P = 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P = 0.0136), LMF (P = 0.0019), and MQ (P = 0.0001). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (P < 0.0001), LMF (P < 0.0001), MQ (P < 0.0001), and QN (P = 0.0026) in wild-type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation significantly increased the in vitro susceptibility to CQ (P = 0.0179) in Pfcrt 76T CQ-resistant parasites.     

Absence of Association between Piperaquine In Vitro Responses and Polymorphisms in the pfcrt,pfmdr1, pfmrp,and pfnhe Genes in Plasmodium falciparum

We have analyzed the profiles of 23 of Plasmodium falciparum strains for their in vitro chemosusceptibilities to piperaquine (PPQ), dihydroartemisinin (DHA), chloroquine, monodesethylamodiaquine, quinine, mefloquine, lumefantrine, atovaquone, pyrimethamine, and doxycycline (DOX) in association with polymorphisms in genes involved in quinoline resistance (Plasmodium falciparum crt [pfcrt], pfmdr1, pfmrp, and pfnhe). The 50% inhibitory concentrations (IC₅₀s) for PPQ ranged from 29 to 98 nM (geometric mean = 57.8 nM, 95% confidence interval [CI] = 51 to 65) and from 0.4 to 5.8 nM for DHA (geometric mean = 1.8 nM, 95% CI = 1.4 to 2.3). We found a significant positive correlation between the responses to PPQ and DHA (r² = 0.17; P = 0.0495) and between the responses to PPQ and DOX (r² = 0.41; P = 0.001). We did not find a significant association between the PPQ IC₅₀ (0.0525 < P < 0.9247) or the DHA IC₅₀ (0.0138 < P < 0.9018) and polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes. There was an absence of cross-resistance with quinolines, and the IC₅₀s for PPQ and DHA were found to be unrelated to mutations in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 transport protein genes, which are involved in quinoline antimalarial drug resistance. These results confirm the interest in and the efficacy of the combination of PPQ and DHA for areas in which parasites are resistant to chloroquine or other quinolines.Over the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine (CQ) and other antimalarial drugs (32). This development has prompted the search for an effective alternative antimalarial drug with minimal side effects. One strategy for reducing the prevalence of malaria is the combinatorial use of drugs, which is thought to protect against the development of resistance to each drug and to reduce the overall rate of transmission of malaria (52). Since 2001, more than 60 countries have officially adopted artemisinin-based combination therapies (ACTs) for the treatment of falciparum malaria (40), and the official first-line antimalarial treatment in Africa is now ACT (18). The artemisinin derivatives cause rapid and effective reductions in the parasite biomass and the level of gametocyte carriage, while the partner drug, which has a longer duration of action, achieves effective clinical and parasitological cure. Several different ACTs have been evaluated, including artesunate-sulfadoxine-pyrimethamine (PY) (50), artesunate-amodiaquine (7), artemether-lumefantrine (LMF) (53), artesunate-mefloquine (MQ) (2), artesunate-chlorproguanil-dapsone (42), artesunate-atovaquone (ATV)-proguanil, artesunate-pyronaridine (44), and dihydroartemisinin (DHA)-piperaquine (PPQ) (1, 22, 47).However, individual P. falciparum isolates resistant to artemisinin in vitro and the first clinical failures have been described in Cambodia (10, 16, 39, 45). In addition, prior therapy with an amodiaquine-containing ACT has been found to select for a reduced response to monodesethylamodiaquine (MDAQ), suggesting that amodiaquine-containing regimens may rapidly lose efficacy in Africa (38). This emergence of parasites resistant to ACTs underlines the fact that novel compounds and combinations must be discovered and developed.DHA-PPQ is an inexpensive, safe, and highly effective treatment for uncomplicated falciparum and vivax malaria (36, 43). DHA-PPQ has been shown to offer a longer posttreatment prophylactic effect following therapy than artemether-lumefantrine (27, 53, 54) or artesunate-amodiaquine (23). The significantly lower risk of recurrent parasitemia after treatment with DHA-PPQ is likely explained by differences in the pharmacokinetics of the nonartemisinin partner drugs. PPQ, a bisquinoline, is estimated to have an elimination half-life of 17 to 33 days (28, 47, 48), while the elimination half-life of lumefantrine is 4 to 10 days (19) and that of amodiaquine is 1 to 6 h (the half-life of its active metabolite, monodesethylamodiaquine, is 1 to 10 days). Bisquinolines are compounds with two quinoline nuclei bound by a covalent aliphatic or aromatic link.The first aim of the present work was to assess the in vitro cross-resistance of PPQ with other quinoline drugs, whether they are artemisinin partners or not. The following drugs were tested: CQ, quinine (QN), MQ, MDAQ, LMF, DHA, ATV, PY, and doxycycline (DOX). The second aim was to identify genetic polymorphisms in the Plasmodium falciparum crt (pfcrt) pfcrt, pfmrp, pfmdr1, and pfnhe-1 genes, which are known to be associated with reduced quinoline susceptibility, that could be associated with decreased susceptibility to PPQ with the goal of identifying molecular markers of PPQ resistance for use in resistance surveillance.     

In Vitro Sensitivity of Plasmodium falciparum Clinical Isolates from the China-Myanmar Border Area to Quinine and Association with Polymorphism in the Na+/H+ Exchanger

Quinine resistance (QNR) in Plasmodium falciparum has been detected in many regions of the world where malaria is endemic. Genetic polymorphisms in at least four genes are implicated in QN susceptibility, and their significance often depends on the genetic background of the parasites. In this study, we have culture-adapted 60 P. falciparum clinical isolates from the China-Myanmar border and assessed their in vitro responses to QN. Our results showed that >50% of the parasite isolates displayed reduced sensitivity to QN, with a half-maximal inhibitory concentration (IC₅₀) above 500 nM. Genotyping of pfcrt found that an overwhelming proportion of the parasite population had the chloroquine-resistant genotype, whereas pfmdr1 mutation genotypes and gene amplification were rare. Genotyping of the P. falciparum Na⁺/H⁺ exchanger gene (pfnhe1) at the minisatellite ms4760 locus identified 10 haplotypes. Haplotype 7, which harbors three copies of the DNNND repeat, was the most predominant, accounting for nearly half of the parasite isolates. Correlation studies did not reveal significant associations of the polymorphisms in pfcrt and pfmdr1 genes with QN response. However, the ms4760 haplotypes were highly associated with in vitro QN responses. In particular, parasite isolates with an increased DNNND copy number tended to have significantly reduced QN susceptibility, whereas parasite isolates with a higher NHNDNHNNDDD copy number had increased QN susceptibility. This study provided further support for the importance of pfnhe1 polymorphisms in influencing QNR in P. falciparum.According to the World Malaria Report 2009, malaria caused an estimated 243 million clinical cases, resulting in nearly 0.9 million deaths, in 2008 (43). While most of the malaria burden is in Africa, it has been estimated that Southeast Asia accounts for 30 and 8% of the global malaria morbidity and mortality, respectively. In the Greater Mekong subregion, malaria epidemiology is characterized by immense geographical heterogeneity in disease distribution with many areas of high endemicity (38). Effective chemotherapy is essential for malaria control, but the emergence and spread of drug resistance in malaria parasites have led to a sharp rise in malaria-related morbidity and mortality (20, 41). This situation is particularly grave in Southeast Asia, where multidrug-resistant (MDR) Plasmodium falciparum poses a major challenge to the control of malaria (39). Therefore, for effective and sustainable malaria management, resistance monitoring and mechanism studies are of high priority, particularly in the era of artemisinin-based combination therapy (42).Quinine (QN) has been a critical antimalarial drug because of its efficacy against chloroquine (CQ)-resistant parasites. In many regions where malaria is endemic, QN is still a primary drug of choice for the treatment of complicated malaria (45). Through its long history in malaria treatment, QN has remained largely effective, and the evolution of QN resistance (QNR) in P. falciparum appears to be slow. However, the observation of reduced sensitivity of P. falciparum to QN in Southeast Asia, South America, and Africa has raised considerable concern (14, 21, 26, 33, 51). In vitro drug assays have found complex patterns of cross-resistance with other quinoline drugs, such as CQ and mefloquine (MQ), suggesting shared resistance mechanisms. Recent genetic and molecular studies indicate that QNR is multifactorial and involves at least four genes: P. falciparum multidrug resistance 1 (pfmdr1), P. falciparum CQ resistance transporter (pfcrt), P. falciparum multidrug resistance-associated protein (pfmrp), and P. falciparum Na⁺/H⁺ exchanger 1 gene (pfnhe1). As its name implies, pfmdr1 is involved in resistance to a number of antimalarials. Global isolates of the parasite show that PfMDR1 harbors a large number of point mutations (11). Genetic studies have found that some PfMDR1 mutations, particularly those that are highly prevalent in South America (S1034C/N1042D/D1246Y), where QN has the longest history of use, are associated with increased QNR (30, 35). In addition, increased copy numbers of pfmdr1 increase resistance not only to MQ (8, 22, 24, 25, 44) but also to other arylamino alcohol drugs, such as QN, halofantrine, and lumefantrine (34). Some mutations in PfCRT, the major CQ resistance (CQR) determinant, are found to be associated with stereo-specific changes in responses to QN and quinidine (6, 10). Recently, a genetic study identified PfMRP as playing a role in the efflux of glutathione, CQ, and QN and contributing to parasite responses to multiple antimalarial drugs (27). In addition to these genes, quantitative trait loci analysis of the Dd2 × HB3 cross further mapped QNR to the pfnhe1 gene, which harbors the minisatellite ms4760 (9). Sequence analysis of laboratory-adapted parasite isolates found that increased copy numbers of the minisatellite repeat (DNNND) are associated with reduced susceptibility to QN (9, 13). Consistent with the role of pfnhe1 in QN response, the Dd2 × HB3 progeny clones with higher levels of QNR also exhibited significantly elevated PfNHE activity (4). Direct evidence of the pfnhe1 involvement in QNR came from transfection studies, where reduced pfnhe1 expression was associated with a significant decrease in QN sensitivity (19). It is noteworthy that the effect of pfnhe1 knockdown is strain specific, providing further support for the complex, multifactorial nature of QNR in P. falciparum. Yet, none of these genes studied so far has accounted for high-level QNR.Southeast Asia has been an epicenter for MDR P. falciparum. Parasites in this region are notorious for their propensity to develop resistance to multiple antimalarial drugs (29). To counter the rapid emergence and spread of drug resistance, malaria drug policies of the countries in Southeast Asia where malaria is endemic have undergone constant changes. CQ and antifolate drugs were abandoned a long time ago (46); resistance to other antimalarial drugs such as MQ has emerged soon after deployment, and QN can no longer be used for malaria monotherapy in this region (17, 26). Although four genes are implicated in reduced QN sensitivity in parasite field isolates, their validity as molecular markers for predicting QNR has been evaluated in only a small number of parasite isolates from diverse regions of the world (13). Therefore, in this study we further investigated the potential association between in vitro QN susceptibility of P. falciparum isolates collected from the China-Myanmar border with genetic polymorphisms in the pfnhe1, pfcrt, and pfmdr1 genes.     

The Polymorphic Linker Domain of pfmdr1 Is Associated with Resistance-Conferring Mutations in Plasmodium falciparum Populations from East and West Africa

Sequence variation in the asparagine/aspartate-rich domain of pfmdr1 in 215 isolates of Plasmodium falciparum from three African countries was compared with published data. The role of this domain in modulating antimalarial sensitivity has not been established. The pfmdr1 86Y allele was significantly associated with different configurations of the Asn/Asp-rich domain in West and East Africa. In Kenya, a specific form of the Asn/Asp-rich domain was significantly linked to the 86Y, 184Y, and 1246Y haplotype of pfmdr1.     

Plasmodium falciparum Polymorphisms Associated with Ex Vivo Drug Susceptibility and Clinical Effectiveness of Artemisinin-Based Combination Therapies in Benin

Artemisinin-based combination therapies (ACTs) are the main option to treat malaria, and their efficacy and susceptibility must be closely monitored to avoid resistance. We assessed the association of Plasmodium falciparum polymorphisms and ex vivo drug susceptibility with clinical effectiveness. Patients enrolled in an effectiveness trial comparing artemether-lumefantrine (n = 96), fixed-dose artesunate-amodiaquine (n = 96), and sulfadoxine-pyrimethamine (n = 48) for the treatment of uncomplicated malaria 2007 in Benin were assessed. pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps polymorphisms were analyzed pretreatment and in recurrent infections. Drug susceptibility was determined in fresh baseline isolates by Plasmodium lactate dehydrogenase enzyme-linked immunosorbent assay (ELISA). A majority had 50% inhibitory concentration (IC₅₀) estimates (the concentration required for 50% growth inhibition) lower than those of the 3D7 reference clone for desethylamodiaquine, lumefantrine, mefloquine, and quinine and was considered to be susceptible, while dihydroartemisinin and pyrimethamine IC₅₀s were higher. No association was found between susceptibility to the ACT compounds and treatment outcome. Selection was observed for the pfmdr1 N86 allele in artemether-lumefantrine recrudescences (recurring infections) (4/7 [57.1%] versus 36/195 [18.5%]), and of the opposite allele, 86Y, in artesunate-amodiaquine reinfections (new infections) (20/22 [90.9%] versus 137/195 [70.3%]) compared to baseline infections. The importance of pfmdr1 N86 in lumefantrine tolerance was emphasized by its association with elevated lumefantrine IC₅₀s. Genetic linkage between N86 and Y184 was observed, which together with the low frequency of 1246Y may explain regional differences in selection of pfmdr1 loci. Selection of opposite alleles in artemether-lumefantrine and artesunate-amodiaquine recurrent infections supports the strategy of multiple first-line treatment. Surveillance based on clinical, ex vivo, molecular, and pharmacological data is warranted.     

Repeat Polymorphisms in the Low-Complexity Regions of Plasmodium falciparum ABC Transporters and Associations with In Vitro Antimalarial Responses

The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC₅₀) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required.     

Reduced Polymorphism in the Kelch Propeller Domain in Plasmodium vivax Isolates from Cambodia

Polymorphism in the ortholog gene of the Plasmodium falciparum K13 gene was investigated in Plasmodium vivax isolates collected in Cambodia. All of them were Sal-1 wild-type alleles except two (2/284, 0.7%), and P. vivax K12 polymorphism was reduced compared to that of the P. falciparum K13 gene. Both mutant allele isolates had the same nonsynonymous mutation at codon 552 (V552I) and were from Ratanak Kiri province. These preliminary data should encourage additional studies for associating artemisinin or chloroquine resistance and K12 polymorphism.     

Atovaquone-Proguanil Remains a Potential Stopgap Therapy for Multidrug-Resistant Plasmodium falciparum in Areas along the Thai-Cambodian Border

Our recent report of dihydroartemisinin-piperaquine failure to treat Plasmodium falciparum infections in Cambodia adds new urgency to the search for alternative treatments. Despite dihydroartemisinin-piperaquine failure, and higher piperaquine 50% inhibitory concentrations (IC₅₀s) following reanalysis than those previously reported, P. falciparum remained sensitive to atovaquone (ATQ) in vitro. There were no point mutations in the P. falciparum cytochrome b ATQ resistance gene. Mefloquine, artemisinin, chloroquine, and quinine IC₅₀s remained comparable to those from other recent reports. Atovaquone-proguanil may be a useful stopgap but remains susceptible to developing resistance when used as blood-stage therapy.     

Temporal and Seasonal Changes of Genetic Polymorphisms Associated with Altered Drug Susceptibility to Chloroquine,Lumefantrine, and Quinine in Guinea-Bissau between 2003 and 2012

In 2008, artemether-lumefantrine was introduced in Guinea-Bissau, West Africa, but quinine has also been commonly prescribed for the treatment of uncomplicated Plasmodium falciparum malaria. An efficacious high-dose chloroquine treatment regimen was used previously. Temporal and seasonal changes of genetic polymorphisms associated with altered drug susceptibility to chloroquine, lumefantrine, and quinine have been described. P. falciparum chloroquine resistance transporter (pfcrt) K76T, pfmdr1 gene copy numbers, pfmdr1 polymorphisms N86Y and Y184F, and pfmdr1 sequences 1034 to 1246 were determined using PCR-based methods. Blood samples came from virtually all (n = 1,806) children <15 years of age who had uncomplicated P. falciparum monoinfection and presented at a health center in suburban Bissau (from 2003 to 2012). The pfcrt K76T and pfmdr1 N86Y frequencies were stable, and seasonal changes were not seen from 2003 to 2007. Since 2007, the mean annual frequencies increased (P < 0.001) for pfcrt 76T (24% to 57%), pfmdr1 N86 (72% to 83%), and pfcrt 76 + pfmdr1 86 TN (10% to 27%), and pfcrt 76T accumulated during the high transmission season (P = 0.001). The pfmdr1 86 + 184 NF frequency increased from 39% to 66% (from 2003 to 2011; P = 0.004). One sample had two pfmdr1 gene copies. pfcrt 76T was associated with a lower parasite density (P < 0.001). Following the discontinuation of an effective chloroquine regimen, probably highly artemether-lumefantrine-susceptible P. falciparum (with pfcrt 76T) accumulated, possibly due to suboptimal use of quinine and despite a fitness cost linked to pfcrt 76T. (The studies reported here were registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00137514","term_id":"NCT00137514"}}NCT00137514 [PSB-2001-chl-amo], {"type":"clinical-trial","attrs":{"text":"NCT00137566","term_id":"NCT00137566"}}NCT00137566 [PSB-2004-paracetamol], {"type":"clinical-trial","attrs":{"text":"NCT00426439","term_id":"NCT00426439"}}NCT00426439 [PSB-2006-coartem], {"type":"clinical-trial","attrs":{"text":"NCT01157689","term_id":"NCT01157689"}}NCT01157689 [AL-eff 2010], and {"type":"clinical-trial","attrs":{"text":"NCT01704508","term_id":"NCT01704508"}}NCT01704508 [Eurartesim 2012].)