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 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.)     

Decreasing pfmdr1 Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is Regaining In Vitro Susceptibility to Mefloquine

Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies—a molecular marker for MQ resistance—in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ.     

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.     

Plasmodium falciparum Na+/H+ Exchanger 1 Transporter Is Involved in Reduced Susceptibility to Quinine

Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC₅₀s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC₅₀ of QN (P = 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC₅₀ of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC₅₀ of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC₅₀ of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC₅₀ of QN (P = 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC₅₀ of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates.Malaria is the most important parasitic disease in the world, affecting 300 to 500 million people and killing 3 million people every year. Quinine (QN) has been used as a malaria treatment for more than 350 years in Africa, with little emergence and spread of resistance. QN remains the first-line antimalarial drug for the treatment of complicated malaria in Europe and Africa. However, despite QN′s efficacy against chloroquine-resistant strains, the emergence of QN resistance (QNR) has been documented. The first cases of QN clinical failure were observed in Brazil and Asia in the 1960s (4, 12). In the 1980s, clinical failures became more frequent in Southeast Asia, South America, and Africa (13, 15, 19, 22, 33). However, QNR is not yet a significant problem. QN remains the first-line drug for severe malaria and remains widely used at present as a second-line therapy for uncomplicated malaria in Africa and other areas. Artemisinin-based combination therapies were proposed as a first-line treatment for uncomplicated malaria 6 years ago. Since 2001, more than 56 countries have officially adopted artemisinin-based combination therapies for the treatment of Plasmodium falciparum malaria. However, individual P. falciparum isolates that are resistant to artemisinin in vitro in Cambodia have been described (14, 21). It is not clear whether these strains are associated with clinical failures. One strategy that health officials can pursue to reduce the prevalence of malaria is to combine QN with other antimalarial drugs such as tetracycline (8, 18) or clindamycin (16).Although some reports of treatment failure of QN exist, it is difficult to fully document QNR because of its short elimination half-life, the requirement to administer the drug three times a day for at least 5 days, drug intolerance often leading to poor compliance, and the lack of reliable data on the correlation between QN 50% inhibitory concentrations (IC₅₀s) and clinical failure. Maximizing the efficacy and longevity of QN as a tool for malaria control will depend critically on pursuing intensive research into identifying in vitro markers as well as implementing in vitro and in vivo surveillance programs such as those championed by the World Antimalarial Resistance Network (30, 31). In this context, there is a need to identify molecular markers that predict QNR and that can provide an active surveillance method to monitor temporal trends in parasite susceptibility (23).QNR appears to share common characteristics with chloroquine resistance. QNR is associated with mutations in both the P. falciparum multidrug resistance gene mdr1 (Pfmdr1) (20, 26) and the chloroquine resistance transporter gene Pfcrt (6, 7, 20). Nevertheless, the mechanism of QNR is still unclear. In addition to Pfmdr1 and Pfcrt, other genetic polymorphisms such as variations in microsatellite length on the sodium/hydrogen exchanger gene Pfnhe-1 (11) and mutations on the multidrug resistance protein gene Pfmrp might contribute to QNR (20). The evidence for the involvement of Pfnhe-1 or Pfmrp in QNR is limited. Only one previous study investigated the association of QN IC₅₀ and polymorphisms in the Pfnhe-1 gene in P. falciparum isolates (11).The objective of the present study was to investigate genetic polymorphisms in Pfcrt, Pfmrp, Pfmdr1, and Pfnhe-1 that could be associated with QNR in order to identify molecular markers of QNR that could be used for surveillance of resistance.     

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

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

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).     

pfcrt polymorphism and chloroquine resistance in Plasmodium falciparum strains isolated in Cambodia

Plasmodium falciparum chloroquine resistance was first detected in Cambodia in the early sixties. Treatment with chloroquine was abandoned 20 years ago. In vitro chloroquine sensitivity monitoring indicates that all eastern Cambodian isolates were sensitive to chloroquine, whereas most isolates collected from western provinces displayed reduced susceptibility to chloroquine. This indicates that the rate of chloroquine resistance remains high and stable in this region in the absence of chloroquine pressure. Characterization of codons 72 to 78 and 218 to 220 of pfcrt revealed six distinct haplotypes, four of which had never been described. The frequency of each haplotype depended on the geographical origin of the samples. The CVIETIF//ISS haplotype was detected in 92% of western Cambodian isolates and in 11% of isolates collected from the eastern province, where CVMNKIF//ISA and CVIDTIF//ISS predominate. The detection of an intermediate haplotype from a susceptible area with 76T/220A, suggests that acquisition of chloroquine resistance might be a stepwise process, during which accumulation of point mutations modulates the response to chloroquine. The association of the K76T mutation with chloroquine resistance was not clear. The mutation was detected in resistant and susceptible samples, suggesting that additional factors are involved in chloroquine resistance. By contrast, the pfcrt D/N75E mutation was strongly associated with the in vitro chloroquine resistance in Cambodian isolates. The N86 allelic form of pfmdr1 was detected in all isolates, consistent with a poor association with resistance to chloroquine. This indicates that in vitro resistance to chloroquine was associated with accumulation of point mutations in pfcrt.     

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.     

Reduced In Vitro Doxycycline Susceptibility in Plasmodium falciparum Field Isolates from Kenya Is Associated with PfTetQ KYNNNN Sequence Polymorphism

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC₅₀s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC₅₀s of >35,000 nM relative to those with IC₅₀s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC₅₀ of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC₅₀ of 9,912 nM (P = 0.0245). Isolates with 1 copy of PftetQ had a median IC₅₀ of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC₅₀ of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC₅₀ of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC₅₀ of 3,352 nM (P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.     

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

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

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.     

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

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

Stearylamine Liposomal Delivery of Monensin in Combination with Free Artemisinin Eliminates Blood Stages of Plasmodium falciparum in Culture and P. berghei Infection in Murine Malaria

The global emergence of drug resistance in malaria is impeding the therapeutic efficacy of existing antimalarial drugs. Therefore, there is a critical need to develop an efficient drug delivery system to circumvent drug resistance. The anticoccidial drug monensin, a carboxylic ionophore, has been shown to have antimalarial properties. Here, we developed a liposome-based drug delivery of monensin and evaluated its antimalarial activity in lipid formulations of soya phosphatidylcholine (SPC) cholesterol (Chol) containing either stearylamine (SA) or phosphatidic acid (PA) and different densities of distearoyl phosphatidylethanolamine-methoxy-polyethylene glycol 2000 (DSPE-mPEG-2000). These formulations were found to be more effective than a comparable dose of free monensin in Plasmodium falciparum (3D7) cultures and established mice models of Plasmodium berghei strains NK65 and ANKA. Parasite killing was determined by a radiolabeled [³H]hypoxanthine incorporation assay (in vitro) and microscopic counting of Giemsa-stained infected erythrocytes (in vivo). The enhancement of antimalarial activity was dependent on the liposomal lipid composition and preferential uptake by infected red blood cells (RBCs). The antiplasmodial activity of monensin in SA liposome (50% inhibitory concentration [IC₅₀], 0.74 nM) and SPC:Chol-liposome with 5 mol% DSPE-mPEG 2000 (IC₅₀, 0.39 nM) was superior to that of free monensin (IC₅₀, 3.17 nM), without causing hemolysis of erythrocytes. Liposomes exhibited a spherical shape, with sizes ranging from 90 to 120 nm, as measured by dynamic light scattering and high-resolution electron microscopy. Monensin in long-circulating liposomes of stearylamine with 5 mol% DSPE-mPEG 2000 in combination with free artemisinin resulted in enhanced killing of parasites, prevented parasite recrudescence, and improved survival. This is the first report to demonstrate that monensin in PEGylated stearylamine (SA) liposome has therapeutic potential against malaria infections.     

Countrywide survey shows very high prevalence of Plasmodium falciparum multilocus resistance genotypes in Cambodia

Cambodia is located in an area of resistance to multiple antimalarials and has been the first country to implement the systematic use of an artesunate-mefloquine combination as first-line treatment for Plasmodium falciparum malaria. Little is known, however, about the prevalence of resistance mutations within the natural parasite populations, impeding rational drug policy in this context. Using direct sequencing of PCR products, we have analyzed sequence polymorphism of the dihydrofolate reductase-thymidylate synthase, dihydropteroate synthetase, and multidrug resistance 1 genes in a large number of clinical P. falciparum isolates collected in various areas of Cambodia. This highlighted a 100% prevalence of haplotypes with multiple mutations in the target genes of antifolates after more than a decade without use of antifolates for malaria therapy. A high prevalence of mutations in Pfmdr1, including mutations associated with decreased in vitro susceptibility to mefloquine and quinine, was also observed. In addition, novel, low-frequency mutations were detected in Pfmdr1. Our findings show an alarming rate of multilocus resistance genotypes in Cambodia, requiring diligent surveillance and imposing limitations on possible future drug combinations.     

Assessment of the Induction of Dormant Ring Stages in Plasmodium falciparum Parasites by Artemisone and Artemisone Entrapped in Pheroid Vesicles In Vitro

The in vitro antimalarial activities of artemisone and artemisone entrapped in Pheroid vesicles were compared, as was their ability to induce dormancy in Plasmodium falciparum. There was no increase in the activity of artemisone entrapped in Pheroid vesicles against multidrug-resistant P. falciparum lines. Artemisone induced the formation of dormant ring stages similar to dihydroartemisinin. Thus, the Pheroid delivery system neither improved the activity of artemisone nor prevented the induction of dormant rings.     

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.     

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.