Surveillance of Artemisinin Resistance in Plasmodium falciparum in India Using the kelch13 Molecular Marker

Malaria treatment in Southeast Asia is threatened with the emergence of artemisinin-resistant Plasmodium falciparum. Genome association studies have strongly linked a locus on P. falciparum chromosome 13 to artemisinin resistance, and recently, mutations in the kelch13 propeller region (Pfk-13) were strongly linked to resistance. To date, this information has not been shown in Indian samples. Pfk-13 mutations were assessed in samples from efficacy studies of artemisinin combination treatments in India. Samples were PCR amplified and sequenced from codon 427 to 727. Out of 384 samples, nonsynonymous mutations in the propeller region were found in four patients from the northeastern states, but their presence did not correlate with ACT treatment failures. This is the first report of Pfk-13 point mutations from India. Further phenotyping and genotyping studies are required to assess the status of artemisinin resistance in this region.     

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.     

Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays

We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.     

Pharmacodynamic Interaction of Doxycycline and Artemisinin in Plasmodium falciparum

Parallel in vitro tests, assessing the inhibition of schizont maturation, were conducted with 31 fresh isolates of Plasmodium falciparum from Thailand, using artemisinin, doxycycline, and combinations of both. The activities of artemisinin and doxycycline are obviously not correlated. Both compounds showed consistent synergism at 50% effective concentration (EC(50)), EC(90), and EC(99) levels.     

Reduced Artemisinin Susceptibility of Plasmodium falciparum Ring Stages in Western Cambodia

The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.     

Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response

Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite''s in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.     

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.)     

Evidence of Selective Sweeps in Genes Conferring Resistance to Chloroquine and Pyrimethamine in Plasmodium falciparum Isolates in India

Treatment of Plasmodium falciparum is complicated by the emergence and spread of parasite resistance to many of the first-line drugs used to treat malaria. Antimalarial drug resistance has been associated with specific point mutations in several genes, suggesting that these single nucleotide polymorphisms can be useful in tracking the emergence of drug resistance. In India, P. falciparum infection can manifest itself as asymptomatic, mild, or severe malaria, with or without cerebral involvement. We tested whether chloroquine- and antifolate drug-resistant genotypes would be more commonly associated with cases of cerebral malaria than with cases of mild malaria in the province of Jabalpur, India, by genotyping the dhps, dhfr, pfmdr-1, and pfcrt genes using pyrosequencing, direct sequencing, and real-time PCR. Further, we used microsatellites surrounding the genes to determine the origins and spread of the drug-resistant genotypes in this area. Resistance to chloroquine was essentially fixed, with 95% of the isolates harboring the pfcrt K76T mutation. Resistant genotypes of dhfr, dhps, and pfmdr-1 were found in 94%, 17%, and 77% of the isolates, respectively. Drug-resistant genotypes were equally likely to be associated with cerebral malaria as with mild malaria. We found evidence of a selective sweep in pfcrt and, to a lesser degree, in dhfr, indicating high levels of resistance to chloroquine and evolving resistance to pyrimethamine. Microsatellites surrounding pfcrt indicate that the resistant genotypes (SVMNT) were most similar to those found in Papua New Guinea.Malaria is arguably the most important vector-borne disease in the world, with annual morbidity and mortality estimates surpassing 300 and 1 million, respectively (20, 61). Over 90% of the total malaria incidence is reported from sub-Saharan and tropical Africa; however, each year Southeast Asia, including the Indian subcontinent, reports approximately 2.5 million malaria cases, 75% of which are from India (20). Further, Plasmodium falciparum incidence in India has increased dramatically over the past few years, including the spread of drug-resistant strains (1, 2, 23, 46, 47).Efforts to control malaria have been hindered by the rapid rise and spread of drug-resistant P. falciparum strains. Chloroquine (CQ)-resistant strains of P. falciparum first appeared in the late 1950s, almost simultaneously in Southeast Asia and South America (51, 58, 64), and subsequently spread through most regions where P. falciparum is endemic. Sulfadoxine-pyrimethamine (SP) was next used as the drug of choice against CQ-resistant malaria; however, resistance quickly emerged on the Thai-Cambodian border around 1980 and is now found throughout most of Southeast Asia, the Amazonian basin of South America, and Africa (1, 2, 7, 17, 40, 53). In India, CQ and SP resistance was first documented in 1973 (42) and 1979 (10), respectively, in the northeast region of the country. Now, studies using molecular markers suggest that CQ and SP resistance is widespread across India (1, 23, 55). However, in India CQ still remains the first line of treatment for Plasmodium vivax malaria and for P. falciparum in low-risk and CQ-sensitive areas. In light of reports of CQ treatment failures, artesunate plus SP (artesunate combination therapy [ACT]) has been introduced in states with high burdens of P. falciparum malaria (6, 45) and is being implemented for other districts with high prevalences of P. falciparum.Resistance to chloroquine has been associated with point mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene (16), while resistance to SP has been linked to the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes (32, 52). Point mutations in P. falciparum multidrug resistance gene 1 (pfmdr-1) have been reported to modulate resistance to different antimalarial drugs, and variations in copy number appear to be associated with mefloquine resistance (12, 36).In order to combat drug resistance in Plasmodium falciparum, it is important to understand the genetic basis and the evolutionary forces affecting loci governing resistance. The discovery of new drug targets and the development of effective drugs and vaccines require careful study of the population genetics of P. falciparum. Investigations regarding point mutations in genes conferring drug resistance and the microsatellite loci that surround these genes can provide information on selection pressures, rates of recombination, and the potential origin of the resistant alleles or mutations.P. falciparum infection can manifest as asymptomatic, mild (uncomplicated) malaria or severe malaria, with or without cerebral involvement; little is known about the factors involved in these clinical manifestations. Cerebral malaria (CM) is one of the most common complications of P. falciparum infection in India, besides severe malaria anemia and multiorgan failure (26, 27, 60, 61). It is not known whether drug-resistant parasites also contribute to the increased risk for CM, especially because patients may receive inadequate treatment with drugs of reduced efficacy. In this context, we were interested in determining whether parasites with resistant genotypes were more often associated with patients diagnosed with CM than with patients with mild malaria (MM). We hypothesized that individuals harboring resistant parasites may be more likely to progress to severe disease due to treatment failure than those with wild-type parasites. Additionally, we wanted to determine whether drug-resistant genotypes in India have evolved locally or have been influenced by gene flow from other regions. To this end, we genotyped four genes associated with drug resistance (pfcrt, dhfr, dhps, and pfmdr-1) and assessed the genetic diversity of microsatellites surrounding pfcrt, dhfr, and dhps from P. falciparum-positive blood samples taken from patients enrolled in a hospital-based study to assess neurological disorders associated with cerebral malaria in central India.     

Plasmodium falciparum Founder Populations in Western Cambodia Have Reduced Artemisinin Sensitivity In Vitro

Reduced Plasmodium falciparum sensitivity to short-course artemisinin (ART) monotherapy manifests as a long parasite clearance half-life. We recently defined three parasite founder populations with long half-lives in Pursat, western Cambodia, where reduced ART sensitivity is prevalent. Using the ring-stage survival assay, we show that these founder populations have reduced ART sensitivity in vitro at the early ring stage of parasite development and that a genetically admixed population contains subsets of parasites with normal or reduced ART sensitivity.     

Potentiation of Artemisinin Activity against Chloroquine-Resistant Plasmodium falciparum Strains by Using Heme Models

The influence of different metalloporphyrin derivatives on the antimalarial activity of artemisinin was studied with two chloroquine-resistant strains of Plasmodium falciparum (FcB1-Colombia and FcM29-Cameroon) cultured in human erythrocytes. This potentiation study indicates that the manganese complex of meso-tetrakis(4-sulfonatophenyl)porphyrin has a significant synergistic effect on the activity of artemisinin against both Plasmodium strains.     

3-Halo Chloroquine Derivatives Overcome Plasmodium falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in P. falciparum

Polymorphism in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was shown to cause chloroquine resistance. In this report, we examined the antimalarial potential of novel 3-halo chloroquine derivatives (3-chloro, 3-bromo, and 3-iodo) against chloroquine-susceptible and -resistant P. falciparum. All three derivatives inhibited the proliferation of P. falciparum; with 3-iodo chloroquine being most effective. Moreover, 3-iodo chloroquine was highly effective at potentiating and reversing chloroquine toxicity of drug-susceptible and -resistant P. falciparum.     

Little Polymorphism at the K13 Propeller Locus in Worldwide Plasmodium falciparum Populations Prior to the Introduction of Artemisinin Combination Therapies

The emergence and spread of artemisinin-resistant Plasmodium falciparum is of huge concern for the global effort toward malaria control and elimination. Artemisinin resistance, defined as a delayed time to parasite clearance following administration of artemisinin, is associated with mutations in the Pfkelch13 gene of resistant parasites. To date, as many as 60 nonsynonymous mutations have been identified in this gene, but whether these mutations have been selected by artemisinin usage or merely reflect natural polymorphism independent of selection is currently unknown. To clarify this, we sequenced the Pfkelch13 propeller domain in 581 isolates collected before (420 isolates) and after (161 isolates) the implementation of artemisinin combination therapies (ACTs), from various regions of endemicity worldwide. Nonsynonymous mutations were observed in 1% of parasites isolated prior to the introduction of ACTs. Frequencies of mutant isolates, nucleotide diversity, and haplotype diversity were significantly higher in the parasites isolated from populations exposed to artemisinin than in those from populations that had not been exposed to the drug. In the artemisinin-exposed population, a significant excess of dN compared to dS was observed, suggesting the presence of positive selection. In contrast, pairwise comparison of dN and dS and the McDonald and Kreitman test indicate that purifying selection acts on the Pfkelch13 propeller domain in populations not exposed to ACTs. These population genetic analyses reveal a low baseline of Pfkelch13 polymorphism, probably due to purifying selection in the absence of artemisinin selection. In contrast, various Pfkelch13 mutations have been selected under artemisinin pressure.     

In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity

Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203 Plasmodium falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms in P. falciparum crt (Pfcrt), Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with the Pfcrt wild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P < 0.0001), and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild-type N allele in Pfmdr-1 codon 86 (93.5%), with only 7 (3.50%) samples with the N86Y mutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Y mutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean of 0.907 ± 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya.     

Origins of the Recent Emergence of Plasmodium falciparum Pyrimethamine Resistance Alleles in Madagascar

The combination of sulfadoxine-pyrimethamine is recommended for use as intermittent preventive treatment of malaria during pregnancy and is deployed in Africa. The emergence and the spread of resistant parasites are major threats to such an intervention. We have characterized the Plasmodium falciparum dhfr (pfdhfr) haplotypes and flanking microsatellites in 322 P. falciparum isolates collected from the Comoros Islands and Madagascar. One hundred fifty-six (48.4%) carried the wild-type pfdhfr allele, 19 (5.9%) carried the S108N single-mutation allele, 30 (9.3%) carried the I164L single-mutation allele, 114 (35.4%) carried the N51I/C59R/S108N triple-mutation allele, and 3 (1.0%) carried the N51I/C59R/S108N/I164L quadruple-mutation allele. Microsatellite analysis showed the introduction from the Comoros Islands of the ancestral pfdhfr triple mutant allele of Asian origin and its spread in Madagascar. Evidence for the emergence on multiple occasions of the I164L single-mutation pfdhfr allele in Madagascar was also obtained. Thus, the conditions required to generate mutants with quadruple mutations are met in Madagascar, representing a serious threat to current drug policy.Despite the increasing financial support for the control of malaria (16, 31), malaria remains a major cause of morbidity and mortality in many developing countries in the tropical world (10). In the Indian Ocean region, where the burden of malaria is restricted to the Comoros Archipelago and Madagascar (33), various intervention strategies are currently being implemented (35). The use of effective and well-tolerated antimalarial drugs is the mainstay of the armory for the control and elimination of Plasmodium falciparum malaria. Artemisinin combination therapies (ACTs) are used for the first-line treatment of P. falciparum infections, and the antifolate sulfadoxine-pyrimethamine (SP) combination is recommended for the intermittent preventive treatment of malaria during pregnancy (IPTp) (3). Indeed, SP, effective in reducing placental malaria and low birth weight, acts as a competitive inhibitor of two enzymes in the parasite''s folate synthesis pathway: dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), respectively (9). Nevertheless, the emergence and the spread of SP-resistant parasites remain major threats that could render this intervention ineffective (19). Amino acid changes at positions 51, 59, 108, and 164 in the P. falciparum dhfr (pfdhfr) gene are strongly associated with pyrimethamine treatment failures (6). Field surveys and experimental studies suggest a stepwise process of accumulation of mutations. The single S108N mutation confers increased in vitro resistance to pyrimethamine (∼20-fold), and subsequent mutations at position 51 (N51I) or 59 (C59R) further increase it (11). Parasites with a triple-mutation allele (51I/59R/108N) have markedly reduced in vitro susceptibility to pyrimethamine, and the presence of the triple-mutation allele increases the risk of SP therapeutic failure. Finally the quadruple-mutation allele, which carries an additional mutation at position 164 (I164L), is highly resistant to pyrimethamine, abrogating the clinical efficacy of SP, as observed in Southeast Asia and South America (26).Recent progress in molecular population genetic studies has greatly facilitated our understanding of the emergence and geographical spread of drug-resistant lineages. In particular, it has been demonstrated that the emergence and dissemination of pyrimethamine-resistant parasites in Africa in the 1990s resulted from the migration of a few resistant mutants from Southeast Asia (29). Indeed, analysis of the microsatellite regions flanking the P. falciparum pfdhfr gene has clearly revealed that in Africa, the pfdhfr triple-mutation allele (I51/R59/N108) associated with pyrimethamine resistance harbored microsatellite haplotypes identical to those found in Southeast Asia (12, 13, 24, 29).In the context of the Indian Ocean, SP resistance has been widely reported in the Comoros Islands (23, 25, 28, 32), whereas SP is still effective in Madagascar (18). However, recent studies performed in Madagascar have shown that the situation is deteriorating and have demonstrated the introduction of P. falciparum multidrug-resistant parasites into Madagascar from the Comoros Islands (17), the rapid rise in the frequency of P. falciparum parasites with both pfdhfr and dhps mutations, and the alarming emergence of the single pfdhfr 164L allele from isolates collected during the last 3 years (2).In order to better understand the origin of the SP-resistant genotypes circulating in the region and determine the importance of gene flow in parasite populations with regard to SP resistance between Africa, the Comoros Islands, and Madagascar, we have characterized the pfdhfr genotype and flanking microsatellite haplotypes of a collection of P. falciparum samples from these areas. Our results confirm that pyrimethamine resistance in Madagascar is essentially related to the introduction from the Comoros Islands of the ancestral pfdhfr triple-mutation allele of Asian origin. Interestingly, however, the I164L single-mutation pfdhfr allele was observed in multiple lineages in areas restricted to the Southeast Madagascar, suggesting local pressure to generate this allele. The coexistence in the same transmission area of mutants with triple mutations and the single I164L mutation indicates that the local emergence of a mutant with quadruple mutations is a likely event that deserves reinforced surveillance.     

The Mu Subunit of Plasmodium falciparum Clathrin-Associated Adaptor Protein 2 Modulates In Vitro Parasite Response to Artemisinin and Quinine

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.     

Resistance of Melanesian elliptocytes (ovalocytes) to invasion by Plasmodium knowlesi and Plasmodium falciparum malaria parasites in vitro.

Erythrocytes from humans with Melanesian elliptocytosis are resistant to invasion by Plasmodium falciparum in vitro and epidemiological evidence suggests they may be resistant to P. vivax and P. malariae. We have examined the ability of P. knowlesi merozoites to invade Melanesian elliptocytes in vitro as a definitive means of examining these cells for resistance to invasion by malarial species with different receptor requirements. The Melanesian elliptocytes were highly resistant to invasion by P. knowlesi merozoites showing that the resistance associated with this erythrocyte variant lies at a level common to the invasion pathway(s) of P. falciparum and P. knowlesi. This makes Melanesian elliptocytosis unique as no other human erythrocyte variant has been shown to be resistant to invasion by both species.