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.     

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.     

Simultaneous Administration of 2-Aminoethyl Diphenylborinate and Chloroquine Reverses Chloroquine Resistance in Malaria Parasites

A nearly complete reversal of chloroquine (CQ) resistance in the CQ-resistant Plasmodium falciparum K-1 strain, with a significant decrease in the mean ± standard deviation (SD) 50% inhibitory concentration (IC₅₀) from 1,050 ± 95 nM to 14 ± 2 nM, was achieved in vitro by the simultaneous administration of 2-aminoethyl diphenylborinate (2-APB). The CQ resistance-reversing activity of 2-APB, which showed the same efficacy as verapamil, was also observed in an in vivo mouse infection model with the CQ-resistant Plasmodium chabaudi AS(30CQ) strain.     

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.)     

Plasmodium vivax Chloroquine Resistance and Anemia in the Western Brazilian Amazon

Data on chloroquine (CQ)-resistant Plasmodium vivax in Latin America is limited, even with the current research efforts to sustain an efficient malaria control program in all these countries where P. vivax is endemic and where malaria still is a major public health issue. This study estimated in vivo CQ resistance in patients with uncomplicated P. vivax malaria, with use of CQ and primaquine simultaneously, in the Brazilian Amazon. Of a total of 135 enrolled subjects who accomplished the 28-day follow-up, parasitological failure was observed in 7 (5.2%) patients, in whom plasma CQ and desethylchloroquine (DCQ) concentrations were above 100 ng/dl. Univariate analysis showed that previous exposure to malaria and a higher initial mean parasitemia were associated with resistance but not with age or gender. In the multivariate analysis, only high initial parasitemia remained significant. Hemoglobin levels were similar at the beginning of the follow-up and were not associated with parasitemia. However, at day 3 and day 7, hemoglobin levels were significantly lower in patients presenting CQ resistance. The P. vivax dhfr (pvdhfr), pvmrp1, pvmdr1, and pvdhps gene mutations were not related to resistance in this small sample. P. vivax CQ resistance is already a problem in the Brazilian Amazon, which could be to some extent associated with the simultaneous report of anemia triggered by this parasite, a common complication of the disease in most of the areas of endemicity.     

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.     

Confirmed Plasmodium vivax Resistance to Chloroquine in Central Vietnam

Plasmodium vivax resistance to chloroquine (CQ) is currently reported in almost all countries where P. vivax is endemic. In Vietnam, despite a first report on P. vivax resistance to chloroquine published in the early 2000s, P. vivax was still considered sensitive to CQ. Between May 2009 and December 2011, a 2-year cohort study was conducted in central Vietnam to assess the recommended radical cure regimen based on a 10-day course of primaquine (0.5 mg/kg/day) together with 3 days of CQ (25 mg/kg). Here we report the results of the first 28-day follow-up estimating the cumulative risk of P. vivax recurrences together with the corresponding CQ blood concentrations, among other endpoints. Out of 260 recruited P. vivax patients, 240 completed treatment and were followed up to day 28 according to the WHO guidelines. Eight patients (3.45%) had a recurrent P. vivax infection, at day 14 (n = 2), day 21 (n = 1), and day 28 (n = 5). Chloroquine blood concentrations, available for 3/8 recurrent infections (days 14, 21, and 28), were above the MIC (>100 ng/ml whole blood) in all of these cases. Fever and parasitemia (both sexual and asexual stages) were cleared by day 3. Anemia was common at day 0 (35.8%), especially in children under 10 years (50%), and hemoglobin (Hb) recovery at day 28 was substantial among anemic patients (median change from day 0 to 28, +1.7 g/dl; interquartile range [IQR], +0.7 to +3.2). This report, based on CQ blood levels measured at the time of recurrences, confirms for the first time P. vivax CQ resistance in central Vietnam and calls for further studies using standardized protocols for accurately monitoring the extent and evolution of P. vivax resistance to chloroquine in Vietnam. These results, together with the mounting evidence of artemisinin resistance in central Vietnam, further highlight the increasing threat of antimalarial drug resistance to malaria elimination in Vietnam.     

Plasmodium falciparum Drug Resistance in Madagascar: Facing the Spread of Unusual pfdhfr and pfmdr-1 Haplotypes and the Decrease of Dihydroartemisinin Susceptibility

The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required.In recent decades, the emergence and subsequent spread of Plasmodium falciparum chloroquine (CQ)- and sulfadoxine-pyrimethamine (SP)-resistant parasites across areas where malaria is endemic have been a challenge to malaria control programs (41, 44). Substantial advances toward gaining an understanding of the genetic basis of antimalarial drug resistance have been made (14). Molecular evolutionary studies have concluded that the CQ-resistant P. falciparum chloroquine resistance transporter (pfcrt) and high-level pyrimethamine-resistant dihydrofolate (pfdhfr) alleles have emerged in a limited number of independent foci, from which they have rapidly spread in the local vicinity and have then invaded areas continent-wide and transferred between continents (1, 36). These lessons of the past have, first, stimulated changes in antimalarial treatment policies by introducing combinations of drugs that act on different targets and, second, resulted in the implementation of effective monitoring systems to detect as early as possible the emergence of resistant parasites on the basis of the assessment of the therapeutic efficacies of antimalarials (25, 46), determination of the decreased sensitivity of the parasites to drugs in vitro (4), and the detection of an increasing prevalence of molecular markers related to drug resistance (24).According to data published from 2002 to 2006, the epidemiological features of P. falciparum CQ and SP resistance differ considerably between Madagascar and the Comoros Islands, two countries located close to each other in the southwestern Indian Ocean (43). In vitro CQ resistance was moderate in Madagascar (29, 33, 45), although the level of therapeutic efficacy was declining. During that time, the rate of CQ resistance was high in the Comoros Islands (22, 23, 30). Likewise, pyrimethamine resistance was absent in Madagascar (28, 32) but was present at high levels in the Comoros Islands (23). The most recent in vivo data obtained on the basis of the WHO 28-day follow-up protocol, conducted in 2006 and 2007 at multiple sites, have confirmed that resistance to all antimalarials except CQ in Madagascar remains rare. Indeed, the prevalence of the clinical failure of treatment with amodiaquine, SP and the combination artesunate and amodiaquine was <5%, while the rate of failure of treatment with CQ was 44% (19). However, the recent demonstration of the introduction of multidrug-resistant P. falciparum parasites into Madagascar from the Comoros Islands (18) and the emergence of the uncommon dihydrofolate reductase I164L genotype in P. falciparum parasites (17) suggest that the situation is currently changing in Madagascar.In this context and in order to help with the rationalization of the malaria elimination policy recently launched by the Malagasy government (withdrawal of CQ in favor of the combination of artesunate plus amodiaquine as first-line treatment and SP usage for intermittent preventive treatment for pregnant women), a large-scale survey was designed and carried out between 2006 and 2008. The aim was to provide a comprehensive spatiotemporal picture of P. falciparum resistance in several geographic areas of Madagascar. We report here the prevalence of P. falciparum parasites harboring mutations correlated with resistance to some quinolines, namely, pfcrt and P. falciparum multidrug resistance gene 1 (pfmdr-1), or SP resistance (pfdhfr, pfdhps) or presenting an increased pfmdr-1 gene copy number, along with the in vitro responses of the parasites to a panel of drugs, including CQ, mefloquine (MF), amodiaquine, quinine (QU), and artemisinin derivatives. In addition, information related to the risk factors that contribute to the spread of antimalarial drug resistance, such as antimalarial resistance in the neighboring islands of the Comoros archipelago, drug pressure, and population movement in Madagascar, was collected (8, 42).     

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.     

Identification and Deconvolution of Cross-Resistance Signals from Antimalarial Compounds Using Multidrug-Resistant Plasmodium falciparum Strains

Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.     

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.     

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).     

Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance.     

Ex Vivo Responses of Plasmodium falciparum Clinical Isolates to Conventional and New Antimalarial Drugs in Niger

Little is known about resistance of Plasmodium falciparum to antimalarials in Sahelian countries. Here we investigated the drug susceptibilities of fresh isolates collected in Niger post-deployment of artemisinin-based combination therapies (ACTs). We found that the parasites remained highly susceptible to new (dihydroartemisinin, lumefantrine, pyronaridine, and piperaquine) and conventional (amodiaquine and chloroquine) antimalarial drugs. The introduction of ACTs in 2005 and their further deployment nationwide have therefore not resulted in a decrease in P. falciparum susceptibilities to these antimalarials.     

Treatment of Murine Cerebral Malaria by Artemisone in Combination with Conventional Antimalarial Drugs: Antiplasmodial Effects and Immune Responses

The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.     

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.     

Prevalence of Plasmodium falciparum Resistance Markers to Sulfadoxine-Pyrimethamine among Pregnant Women Receiving Intermittent Preventive Treatment for Malaria in Uganda

The aim of this study was to assess the prevalence of mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes among pregnant women using sulfadoxine-pyrimethamine (SP) as an intermittent preventive treatment (IPTp). A molecular epidemiological study of P. falciparum parasite resistance markers to SP was conducted from August 2010 to February 2012 in Mukono district in central Uganda. DNA was extracted from 413 P. falciparum-positive samples. Real-time PCR, followed by melting curve analysis, was used to characterize point mutations in the Pfdhfr and Pfdhps genes that are associated with SP resistance. The prevalence of the single-nucleotide mutations in Pfdhfr at codons 51I, 59R, and 108N and in Pfdhps at codons 437G and 540E was high (>98%), reaching 100% fixation after one dose of SP, while the prevalence of 581G was 3.3% at baseline, reaching 12.5% after one dose of SP. At baseline, the prevalence of Pfdhfr and Pfdhps quintuple mutations was 89%, whereas the sextuple mutations (including 581G) were not prevalent (3.9%), reaching 16.7% after one dose of SP. However, the numbers of infections at follow-up visits were small, and hence there was insufficient statistical power to test whether there was a true rise in the prevalence of this allele. The overall high frequency of Pfdhfr and Pfdhps quintuple mutations throughout pregnancy excluded further analyses of possible associations between certain haplotypes and the risk of lower birth weight and anemia. However, women infected with P. falciparum had 1.3-g/dl-lower hemoglobin levels (P = 0.001) and delivered babies with a 400-g-lower birth weight (P = 0.001) compared to nonparasitemic women. Despite this, 44 women who were P. falciparum positive at baseline became negative after one or two doses of SP (i.e., 50.5%), implying that SP-IPTp still has some efficacy. P. falciparum resistance markers to SP are high in this population, whereas P. falciparum infection was associated with poor birth outcomes.     

Chloroquine resistance-conferring mutations in pfcrt give rise to a chloroquine-associated H+ leak from the malaria parasite's digestive vacuole

Chloroquine resistance in the malaria parasite Plasmodium falciparum is conferred by mutations in the P. falciparum chloroquine resistance transporter (PfCRT). PfCRT localizes to the membrane of the parasite's internal digestive vacuole, an acidic organelle in which chloroquine accumulates to high concentrations and exerts its toxic effect. Mutations in PfCRT are thought to reduce chloroquine accumulation in this organelle. How they do so is the subject of ongoing debate. Recently we have shown that in the presence of chloroquine there is an increased leak of H⁺ from the digestive vacuole in chloroquine-resistant but not chloroquine-sensitive parasites. Here, using transfectant parasite strains of a single genetic background and differing only in their pfcrt allele, we show that chloroquine resistance-conferring PfCRT mutations are responsible for this chloroquine-associated H⁺ leak. This is consistent with the hypothesis that the chloroquine resistance-conferring forms of PfCRT mediate the efflux of chloroquine, in association with H⁺, from the malaria parasite's digestive vacuole.     

Origin and Dissemination across the Colombian Andes Mountain Range of Sulfadoxine-Pyrimethamine Resistance in Plasmodium falciparum

The therapeutic efficacy of sulfadoxine-pyrimethamine (SP) in treating uncomplicated Plasmodium falciparum malaria is unevenly distributed in Colombia. The Andes mountain range separates regions in the west where malaria is endemic from those in the east and constitutes a barrier against gene flow and the dispersal of parasite populations. The distribution of dhfr and dhps genotypes of 146 P. falciparum samples from the eastern Amazon and Orinoco basins and Northwest and Southwest Pacific regions of Colombia was consistent with the documented levels of therapeutic efficacy of SP. The diversity of four dhfr- and dhps-linked microsatellites indicated that double- and triple-mutant alleles for both resistance loci have a single origin. Likewise, multilocus association genotypes, including two unlinked microsatellite loci, suggested that genetic exchanges between the eastern Orinoco and Northwest Pacific populations has taken place across the Andes, most probably via migration of infected people.Enzymes involved in folate metabolism are targeted by the anifolate antimalarial drugs. Pyrimethamine targets the enzyme dihydrofolate reductase (DHFR) and, in combination with sulfadoxine, which targets the enzyme dihydropteroate synthase (DHPS), has been widely used as first-line treatment for uncomplicated Plasmodium falciparum malaria worldwide. In South America, pyrimethamine was introduced and used as a mass treatment in the 1950s in Venezuela (10) until drug-resistant cases were detected (16). By 1968, pyrimethamine-resistant/sulfadiazine-sensitive parasites were documented in Brazil, Venezuela, and Colombia (28). The sulfadoxine-pyrimethamine (SP) combination was introduced in parts of South America in the 1970s and was used until 1981 in Colombia as an alternative to chloroquine, to which resistance was widespread (8). Soon after its introduction, treatment failure was reported, and resistance rapidly disseminated in the Amazon and Orinoco basins (12). In Colombia, SP resistance is unevenly distributed. High SP resistance levels (80%) have been consistently reported from the Colombian Amazon basin (21), while moderate levels (6% to 24%) are observed in the Caribbean, the Cauca Valley, and northwestern regions (3, 4). This contrasts with regions on the southern Pacific coast where the SP combination is still efficacious (13).P. falciparum resistance to pyrimethamine is acquired by the progressive accumulation of mutations at the dhfr locus. The S108N substitution is initially required for the acquisition of the resistant phenotype; in this genetic context, resistance increases with the accumulation of additional mutations at position N51I or C59R, and still higher levels of resistance are reached with the further acquisition of mutations at positions C50R and I164L (7, 14, 15). Resistance to sulfadoxine also depends on the progressive accumulation of mutations in the dhps locus at codons 436, 437, 540, 581, and 613. The A437G mutation is initially required and is followed by mutations at codons A581G, S436A, K540E, and A613S, which confer incrementally higher levels of resistance (14).The current pattern of P. falciparum SP resistance in Colombia raises questions as to the nature, frequency, and origin of the circulating dhfr and dhps genotypes. Analysis of microsatellite markers linked to dhfr and dhps has shown that some mutant resistance alleles in Asia and Africa have become globally dispersed (27), while unlinked polymorphic loci on different chromosomes have suggested that the clonal expansion of a few resistant parasite genotypes underlies the spread of drug resistance in South America (6). Interestingly, in Colombia, the Andes mountain range separates the Pacific coast in the west from the Amazon and Orinoco basins in the east and may constitute a barrier against genetic exchanges. Here, we analyze the genetic nature and origin of P. falciparum SP resistance in Colombia. The results will help to the design strategies to prevent the spread of drug resistance in Colombia and eventually other Andean countries.