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.     

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).     

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.     

Plasmodium falciparum kelch 13: a potential molecular marker for tackling artemisinin-resistant malaria parasites

Although artemisinin combination therapies have been deployed as a first-line treatment for uncomplicated malaria in almost all endemic countries, artemisinin-resistant parasites have emerged and have gradually spread across the Greater Mekong subregions. There is growing concern that the resistant parasites may migrate to or emerge indigenously in sub-Saharan Africa, which might provoke a global increase in malaria-associated morbidity and mortality. Therefore, development of molecular markers that enable identification of artemisinin resistance with high sensitivity is urgently required to combat this issue. In 2014, a potential artemisinin-resistance responsible gene, Plasmodium falciparum kelch13, was discovered. Here, we review the genetic features of P. falciparum kelch13 and discuss its related resistant mechanisms and potential as a molecular marker.     

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.     

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.     

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.     

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.     

Molecular Correlates of High-Level Antifolate Resistance in Rwandan Children with Plasmodium falciparum Malaria

Antifolate drugs have an important role in the treatment of malaria. Polymorphisms in the genes encoding the dihydrofolate reductase and dihydropteroate synthetase enzymes cause resistance to the antifol and sulfa drugs, respectively. Rwanda has the highest levels of antimalarial drug resistance in Africa. We correlated the efficacy of chlorproguanil-dapsone plus artesunate (CPG-DDS+A) and amodiaquine plus sulfadoxine-pyrimethamine (AQ+SP) in children with uncomplicated malaria caused by Plasmodium falciparum parasites with pfdhfr and pfdhps mutations, which are known to confer reduced drug susceptibility, in two areas of Rwanda. In the eastern province, where the cure rates were low, over 75% of isolates had three or more pfdhfr mutations and two or three pfdhps mutations and 11% had the pfdhfr 164-Leu polymorphism. In the western province, where the cure rates were significantly higher (P < 0.001), the prevalence of multiple resistance mutations was lower and the pfdhfr I164L polymorphism was not found. The risk of treatment failure following the administration of AQ+SP more than doubled for each additional pfdhfr resistance mutation (odds ratio [OR] = 2.4; 95% confidence interval [CI] = 1.01 to 5.55; P = 0.048) and each pfdhps mutation (OR = 2.1; 95% CI = 1.21 to 3.54; P = 0.008). The risk of failure following CPG-DDS+A treatment was 2.2 times higher (95% CI = 1.34 to 3.7) for each additional pfdhfr mutation, whereas there was no association with mutations in the pfdhps gene (P = 0.13). The pfdhfr 164-Leu polymorphism is prevalent in eastern Rwanda. Antimalarial treatments with currently available antifol-sulfa combinations are no longer effective in Rwanda because of high-level resistance.Antifolate drugs have an important role in the treatment of Plasmodium falciparum malaria and are among the most widely available drugs. Antifols and sulfonamides target the de novo folate synthesis pathway of the parasite, inhibiting the activities of two key enzymes, dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), respectively, and thereby interrupting DNA synthesis. When they are used in combinations, DHFR and DHPS inhibitors act synergistically. Sulfadoxine-pyrimethamine (SP) is the most commonly used antifolate for the treatment of malaria, but its efficacy varies widely because of resistance, which has spread rapidly in many regions where malaria is endemic. Antifolate drug resistance results from polymorphisms in the genes encoding the DHFR and DHPS enzymes. Mutations in the pfdhfr gene and in the corresponding gene in Plasmodium vivax are acquired sequentially, with each mutation conferring a stepwise reduction in susceptibility (11, 12). This progression is less clearly delineated for mutations in the pfdhps gene. Under drug pressure, antifolate resistance can be selected rapidly both in vivo and in vitro (17, 24).The relative importance of different mutation patterns in predicting therapeutic responses remains a subject of debate, particularly when there is significant background immunity to augment antimalarial drug effects (11, 15). In Africa, treatment failure following SP treatment of falciparum malaria is often attributed to mutations at codons 108, 51, and 59 of the pfdhfr gene product (referred to as the triple mutant, which has Asn-108/Ile-51/Arg-59 mutations) and point mutations at codon 437 and/or 540 of the pfdhps gene product (referred to as the double mutant, which has Gly-437/Glu-540 mutations) (14, 29). A significantly higher level of resistance to antifolates is associated with a mutation at codon 164 of the pfdhfr gene product (Ile instead of Leu). This almost invariably occurs in addition to the mutations at codons 108, 51, and 59; and thus, parasites with the 164-Leu mutation are usually called quadruple mutants (10, 25). Until recently, quadruple mutants were confined to parts of Asia and South America (16). In 1997, Watkins et al. (30) predicted that selection of this fourth mutation would compromise the clinical usefulness of the more potent antifolate combination chlorproguanil-dapsone (CPG-DDS), designed primarily for use in Africa. East Africa has historically had more resistant P. falciparum parasites than other areas of Africa. The resistance patterns in that region often act as harbingers of the spread of resistance to the remainder of the continent.Between 2001 and 2006, various artemisinin-based combination treatments were tested in Rwanda to identify the best treatment for uncomplicated malaria to be adopted as the first-line therapy in that country (8, 13, 26, 27). In 2006, the efficacy and safety of CPG-DDS plus artesunate (CPG-DDS+A) were compared with the efficacy and safety of amodiaquine plus SP (AS+SP) (6, 7).The aim of the present study was to evaluate the clinical efficacies of these two antifolate combinations, CPG-DDS+A and AQ+SP, in relation to drug resistance mutations in the pfdhfr and pfdhps genes in Rwanda, a country with high levels of antimalarial drug resistance. Although neither combination is now used in Rwanda, at the time we carried out this study, SP was still recommended for the intermittent preventive treatment of malaria (IPT) during pregnancy and other antifolates had been suggested to be possible treatments for malaria in Rwanda (28).     

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.     

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.)     

Prevalence of Polymorphisms in Antifolate Drug Resistance Molecular Marker Genes pvdhfr and pvdhps in Clinical Isolates of Plasmodium vivax from Kolkata,India

Sulfadoxine-pyrimethamine has never been recommended for the treatment of Plasmodium vivax malaria as the parasite is intrinsically resistant to pyrimethamine. The combination was introduced as a promising agent to treat Plasmodium falciparum malaria in many countries but was withdrawn after a few years due to development and spread of resistant strains. Presently, sulfadoxine-pyrimethamine is used as a partner drug of artemisinin-based combination therapy to treat uncomplicated falciparum malaria, and a combination of artesunate-sulfadoxine-pyrimethamine is currently in use in India. In countries like India, where both P. vivax and P. falciparum are equally prevalent, some proportion of P. vivax bacteria is exposed to sulfadoxine-pyrimethamine due to misdiagnosis and mixed infections. As reports on the in vivo therapeutic efficacy of sulfadoxine-pyrimethamine in P. vivax are rare, the study of mutations in the marker genes P. vivax dhfr (pvdhfr) and pvdhps is important for predicting drug selection pressure and sulfadoxine-pyrimethamine resistance monitoring. We studied the prevalence of point mutations and haplotypes of both the genes in 80 P. vivax isolates collected from urban Kolkata, India, by the DNA sequencing method. Point mutation rates in both the genes were low. The double mutant pvdhfr A₁₅N₅₀R₅₈N₁₁₇I₁₇₃ (mutations are in boldface) and the single mutant pvdhps genotype S₃₈₂G₃₈₃K₅₁₂A₅₅₃V₅₈₅ were more prevalent, while 35% of the isolates harbored the wild-type genotype. The triple mutant ANRNI-SGKAV was found in 29.9% isolates. No quintuple mutant genotype was recorded. The P. vivax parasites in urban Kolkata may still be susceptible to sulfadoxine-pyrimethamine. Hence, a combination of antimalarial drugs like artesunate-sulfadoxine-pyrimethamine introduced for P. falciparum infection might be effective in P. vivax infection also. Study of the therapeutic efficacy of this combination in P. vivax is thus strongly suggested. (The study protocol was registered in the Clinical Trial Registry-India [CTRI] of the Indian Council of Medical Research under registration number CTRI/2011/09/002031.)     

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.     

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.     

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.     

Characteristics of Genetic Hitchhiking around Dihydrofolate Reductase Gene Associated with Pyrimethamine Resistance in Plasmodium falciparum Isolates from India

Sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum has been widespread across continents, causing the major hurdle of controlling malaria. Resistance is encoded mainly by point mutations in P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) target genes. To study the origin and evolution of pyrimethamine resistance on the Indian subcontinent, microsatellite markers flanking the pfdhfr gene were mapped. Here we describe the characteristics of genetic hitchhiking around the pfdhfr gene among 190 P. falciparum isolates. These isolates were collected from five different geographical regions of India (Uttar Pradesh, Madhya Pradesh, Assam, Orissa, and Andaman and Nicobar Islands) where malarial transmission rates and levels of drug resistance vary across regions. Among the isolates, we observed a significant reduction in genetic variation in the ±20-kb vicinity of the mutant pfdhfr alleles due to hitchhiking. This reduction in genetic diversity was more prominent around quadruple pfdhfr alleles (heterozygosity [H_e] = 0.23) than around double (H_e = 0.365) and single (H_e = 0.465) mutant alleles. Asymmetry in the selective sweep flanking the pfdhfr alleles was observed with regional isolates, emphasizing the drug usage with the parasite population. All the pfdhfr alleles share a single microsatellite haplotype and seem to have originated from a single progenitor similar to that of Southeast Asian (Thailand) pfdhfr mutants. Results of the present study also indicate that the emergence of drug-resistant alleles is a recent phenomenon in India compared to Southeast Asian countries.Malaria is the major cause of death in tropical and subtropical areas. Among all the four species of Plasmodium, P. falciparum causes the most severe form of malaria, which often leads to death. In the absence of effective vaccines, management of the disease is dependent largely on antimalarial drugs. However, the parasite has developed resistance to most commonly used antimalarial drugs, thus posing a major problem for malaria control programs (14). Emergence of chloroquine resistance in India has required the use of alternative antimalarials, like the sulfadoxine-pyrimethamine (SP) combination, in the recent past. However, the current drug policy in India for treating uncomplicated P. falciparum malaria cases in highly chloroquine-resistant areas prescribes the use of artemisinin combination therapy, consisting of artesunate plus SP (www.nvbdcp.gov.in). Thus, SP drug pressure on the parasite population continues to be high in the country.SP acts synergistically, targeting the enzymes dihydropteroate synthase and dihydrofolate reductase, which are involved in the folate biosynthesis pathway of the parasite (13). These parasite enzymes and molecular mechanisms underlying antifolate drug resistance have been well characterized (12, 30, 36). Sequencing of the pfdhfr gene from pyrimethamine-resistant and -sensitive parasite isolates identified the important amino acid changes that alter the binding of the drug and hence confer the resistance (12, 30). A Ser-to-Asn change at amino acid position 108 in P. falciparum dihydrofolate reductase is the key event in the development of the resistance and additional mutations at amino acid residues 51, 59, and 164, further increasing the tolerance of the parasite toward the drug (35).Levels of pyrimethamine resistance differ among the continental population of the parasite due to the presence of various pfdhfr mutant alleles. The triple mutant A₁₆I₅₁R₅₉N₁₀₈I₁₆₄ pfdhfr allele is present mostly in Africa and Southeast Asia, while the quadruple mutant A₁₆I₅₁R₅₉N₁₀₈L₁₆₄ allele is observed predominantly in Southeast Asia (6, 26). Previously we have reported the A₁₆N₅₁C₅₉N₁₀₈I_164, A₁₆N₅₁R₅₉N₁₀₈I_164, A₁₆I₅₁R₅₉N₁₀₈I_164, A₁₆N₅₁R₅₉N₁₀₈L_164, and A₁₆I₅₁R₅₉N₁₀₈L₁₆₄ pfdhfr mutant alleles from India (1-3). Two different pfdhfr triple mutant R₅₀I₅₁C₅₉N₁₀₈I₁₆₄ and C₅₀I₅₁C₅₉N₁₀₈L₁₆₄ alleles were found in South America (8, 31). The occurrence of Bolivia repeats was found exclusively in South America, which suggested two different evolutionary lineages of dhfr in South America (8, 24).Microsatellites are simple sequence repeats, abundantly present in the P. falciparum genome, and polymorphism in the number of repeats occurs mainly because of strand slippage during DNA replication (9, 15, 18, 19). These markers, located close to the resistance loci, have been employed to study the emergence and spread of SP resistance alleles in the P. falciparum population (11). There are reports which revealed that the triple mutant pfdhfr allele has three independent origins, one from Southeast Asia (26, 27, 33) and two from South America (8, 23, 24). A majority of the triple mutant alleles found in Africa originated and migrated from Southeast Asia (7, 21, 22, 25, 33). However, minor, independent origins of the triple mutant dhfr allele have been observed (7, 22). Southeast Asian triple and quadruple pfdhfr mutants have a single origin, whereas the African double mutant originated twice (26, 27, 32).Due to continued drug pressure, the antifolate drug resistance-associated pfdhfr mutations have been reported from India (1-3). However, the spread of SP resistance alleles across the Indian subcontinent has not been investigated. Therefore, in the present study, we determined the microsatellite haplotypes flanking pfdhfr in isolates from five different geographical regions of India (Uttar Pradesh [UP], Madhya Pradesh [MP], Assam, Orissa, and Andaman and Nicobar Islands) having different malaria transmission dynamics and drug resistance levels (Fig. ​(Fig.1).1). It has been known that the northern state of UP has low levels of malaria transmission and drug resistance, whereas the central state of MP has moderate levels of malaria transmission and drug resistance. On the other hand, the northeastern state of Assam, eastern state of Orissa, and Andaman and Nicobar Islands have high levels of malaria transmission and drug resistance (17). Previously, we reported the presence of different pfdhfr genotypes among isolates from these different regions, which seem to show a relationship to malaria transmission intensities in these areas (2, 3). For example, the quadruple and triple pfdhfr mutations were present only among isolates from the regions where the intensity of malaria transmission is high, i.e., from Andaman and Nicobar Islands, Assam, and Orissa, but not from the low-malaria-transmission area of UP. We report here that the impact levels of selection on hitchhiking differ among the regions of high and low selection at pfdhfr and that there seems to be an independent origin of the pfdhfr mutant alleles in India.Open in a separate windowFIG. 1.Map of India showing sample collection sites.     

Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected with Plasmodium falciparum Malaria Parasites

In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.