Defining the Role of Mutations in Plasmodium vivax Dihydrofolate Reductase-Thymidylate Synthase Gene Using an Episomal Plasmodium falciparum Transfection System

Plasmodium vivax resistance to antifolates is prevalent throughout Australasia and is caused by point mutations within the parasite dihydrofolate reductase (DHFR)-thymidylate synthase. Several unique mutations have been reported in P. vivax DHFR, and their roles in resistance to classic and novel antifolates are not entirely clear due, in part, to the inability to culture P. vivax in vitro. In this study, we use a homologous system to episomally express both wild-type and various mutant P. vivax dhfr (pvdhfr) alleles in an antifolate-sensitive line of P. falciparum and to assess their influences on the susceptibility of the recipient P. falciparum line to commonly used and new antifolate drugs. Although the wild-type pvdhfr-transfected P. falciparum line was as susceptible to antifolate drugs as the P. falciparum parent line, the single (117N), double (57L/117T and 58R/117T), and quadruple (57L/58R/61M/117T) mutant pvdhfr alleles conferred a marked reduction in their susceptibilities to antifolates. The resistance index increased with the number of mutations in these alleles, indicating that these mutations contribute to antifolate resistance directly. In contrast, the triple mutant allele (58R/61M/117T) significantly reversed the resistance to all antifolates, indicating that 61M may be a compensatory mutation. These findings help elucidate the mechanism of antifolate resistance and the effect of existing mutations in the parasite population on the current and new generation of antifolate drugs. It also demonstrates that the episomal transfection system has the potential to provide a rapid screening system for drug development and for studying drug resistance mechanisms in P. vivax.Of the four species of Plasmodium that commonly cause malaria in humans, Plasmodium vivax is the most widely distributed and can account for up to 80 million cases annually (25). Although P. vivax infections cause less mortality than P. falciparum, they do cause a debilitating disease that contributes to significant morbidity and economic loss in many regions where P. vivax is endemic (25). This is compounded by frequent relapses that can occur many times and for many months after the initial infection (6, 16, 22).Plasmodium vivax parasites are susceptible to most antimalarial drugs. However, over the last 20 years there have been many reports that highlight the significant increase in resistance of P. vivax malaria to chloroquine, the recommended first-line treatment of P. vivax, and/or sulfadoxine-pyrimethamine (SP) (1, 7, 9, 13, 29, 31, 41, 42). Although the determinant(s) for chloroquine resistance in P. vivax remains elusive, a genetic basis for antifolate resistance in P. vivax has been identified as polymorphisms in the P. vivax dihydrofolate reductase (PvDHFR) active site, the same mechanism observed in antifolate resistance in P. falciparum (2, 11, 12, 17, 18, 20, 21, 24, 39).In P. falciparum, point mutations within DHFR have been determined as the cause for antifolate resistance, such as pyrimethamine and cycloguanil resistance (4, 5, 8, 30, 32-36, 44, 45). It has been shown that resistance to antifolates results from the accumulation of mutations in the P. falciparum DHFR, principally A16V, N51I, C59R, S108N or S108T, and I164L.A large number of point mutations have been identified in PvDHFR, and some were reported to be prevalent in many areas of P. vivax endemicity (2, 12, 18, 20, 21, 39). A particular set of mutations (F57L + S58R + T61M + S117T) within the P. vivax DHFR was shown to correlate with SP treatment failures (18, 39) and to confer significant antifolate resistance when transfected into antifolate-sensitive P. falciparum (28). Resistance is due to alterations to the pyrimethamine binding site of PvDHFR that reduces parasite-drug interactions (23, 28). However, the contribution of these PvDHFR mutations to resistance to a variety of antifolates drugs is not clear.Due to the difficulty in maintaining P. vivax in in vitro cultures, most studies of the mutations within PvDHFR have been limited to surrogate biological systems such as yeast and Escherichia coli (17, 19, 24, 38). Although these systems have obvious experimental utility, they are different from Plasmodium in many biological aspects, particularly in membrane structures and transporters that can potentially affect the susceptibility to drugs. A recent publication (28) showed that the pvdhfr-ts quadruple mutant allele (57L + 58R + 61M + 117T) that is episomally expressed in P. falciparum provides significant protection against antifolates. This demonstrated an excellent potential of using P. falciparum as a biological system for the transgenic expression of pvdhfr-ts alleles to assess DHFR-TS interactions with antifolates.We report here the use of this P. falciparum expression system to assess the effect that specific mutations within the P. vivax DHFR have on conventional and new-generation antifolate drugs. The findings improve our understanding of the effect of various mutant pvdhfr alleles observed in the field on the parasite responses to current and new generations of antifolates, improve the prediction of malaria drug treatment outcome, and provide a useful tool for drug development.     

In Vitro Activity of Pyronaridine against Multidrug-Resistant Plasmodium falciparum and Plasmodium vivax

Pyronaridine, a Mannich base antimalarial, has demonstrated high in vivo and in vitro efficacy against chloroquine-resistant Plasmodium falciparum. Although this drug has the potential to become a prominent artemisinin combination therapy, little is known about its efficacy against drug-resistant Plasmodium vivax. The in vitro antimalarial susceptibility of pyronaridine was assessed in multidrug-resistant P. vivax (n = 99) and P. falciparum (n = 90) isolates from Papua, Indonesia, using a schizont maturation assay. The median 50% inhibitory concentration (IC₅₀) of pyronaridine was 1.92 nM (range, 0.24 to 13.8 nM) against P. falciparum and 2.58 nM (range, 0.13 to 43.6 nM) against P. vivax, with in vitro susceptibility correlating significantly with chloroquine, amodiaquine, and piperaquine (r_s [Spearman''s rank correlation coefficient] = 0.45 to 0.62; P < 0.001). P. falciparum parasites initially at trophozoite stage had higher IC₅₀s of pyronaridine than those exposed at the ring stage (8.9 nM [range, 0.6 to 8.9 nM] versus 1.6 nM [range, 0.6 to 8.9 nM], respectively; P = 0.015), although this did not reach significance for P. vivax (4.7 nM [range, 1.4 to 18.7 nM] versus 2.5 nM [range, 1.4 to 15.6 nM], respectively; P = 0.085). The excellent in vitro efficacy of pyronaridine against both chloroquine-resistant P. vivax and P. falciparum highlights the suitability of the drug as a novel partner for artemisinin-based combination therapy in regions where the two species are coendemic.Almost 40% of the world''s population is at risk for infection by Plasmodium vivax, with an estimated 132 to 391 million clinical infections each year (19). Although chloroquine (CQ) remains the treatment of choice in most of the P. vivax-endemic world, this status is now being undermined by the emergence and spread of chloroquine-resistant (CQR) P. vivax. First reported in the 1980s on the island of New Guinea (2, 23), CQR P. vivax has since spread to other parts of Asia and recently to South America (1). In Papua, Indonesia, CQ resistance in P. vivax has reached levels precluding the use of CQ in most of the province (22, 30). There is an urgency to assess the efficacies of alternative antimalarial agents against drug-resistant P. vivax and to develop new strategies to combat the parasite.Pyronaridine (Pyr), a Mannich base synthesized in China in the 1970s (3, 16), is being developed as a novel antimalarial for multidrug-resistant malaria. It demonstrates potent in vitro activity against erythrocytic stages of Plasmodium falciparum (8, 24, 26, 36), retaining efficacy against CQR isolates (12, 17, 18). Clinical trials have shown excellent efficacy of monotherapy against multidrug-resistant falciparum malaria (14, 24, 25), with the early therapeutic response faster when combined with artesunate (20). Phase III studies with a coformulation of Pyramax (Shin Poong Pharmaceuticals) containing artesunate plus pyronaridine have recently been completed (34).Less is known of the antimalarial properties of pyronaridine against P. vivax, although early clinical studies in China demonstrated a rapid therapeutic response (3). To investigate the activity of pyronaridine against CQR P. vivax, we applied a modified schizont maturation assay on fresh field isolates from Papua, Indonesia, where CQR P. vivax is highly prevalent.     

CD68 acts as a major gateway for malaria sporozoite liver infection

After being delivered by the bite from an infected mosquito, Plasmodium sporozoites enter the blood circulation and infect the liver. Previous evidence suggests that Kupffer cells, a macrophage-like component of the liver blood vessel lining, are traversed by sporozoites to initiate liver invasion. However, the molecular determinants of sporozoite–Kupffer cell interactions are unknown. Understanding the molecular basis for this specific recognition may lead to novel therapeutic strategies to control malaria. Using a phage display library screen, we identified a peptide, P39, that strongly binds to the Kupffer cell surface and, importantly, inhibits sporozoite Kupffer cell entry. Furthermore, we determined that P39 binds to CD68, a putative receptor for sporozoite invasion of Kupffer cells that acts as a gateway for malaria infection of the liver.Malaria remains one of the world’s most devastating diseases, afflicting close to 500 million people and causing nearly 1 million deaths every year. Parasite resistance to drugs is of major concern (White et al., 2014), and new drug targets need to be urgently identified. Some progress has recently been made in malaria vaccine development, but identification of new vaccine targets remains a high priority (Moorthy et al., 2004; Moorthy and Kieny, 2010). A better understanding of parasite infection of the human host is crucial for the development of new tools to fight the disease.Infection of a vertebrate host is initiated by the bite of an infected female mosquito. Sporozoites released with the mosquito saliva enter the blood circulation and exit in the liver to establish a productive infection. Hepatocyte infection leads to a dramatic amplification of parasite numbers: 1 sporozoite generates up to 10,000 merozoites that are subsequently released into the bloodstream where they continuously propagate inside red blood cells, causing disease symptoms (Sturm et al., 2006). The pre-erythrocytic liver stages represent a severe bottleneck in parasite numbers and constitute a prime target for induction of sterile immunity. Understanding the mechanisms of parasite liver invasion may provide crucial insights for pre-erythrocytic malaria drug and vaccine development.After delivery by an infected mosquito, sporozoites circulate through the entire body. What cues does the parasite use to exit the blood circulation in the liver and which mechanisms operate for sporozoite exit from the circulation are fundamental questions that are incompletely understood. The liver has specialized blood vessels, the sinusoids, whose walls are made up by two cell types: fenestrated endothelial cells and macrophage-like Kupffer cells (Widmann et al., 1972). Circulating sporozoites are believed to be captured via strong interaction between circumsporozoite protein (CSP), a major sporozoite surface protein, and the highly sulfated heparan sulfate proteoglycans (HSPGs) that are synthesized by stellate cells in the space of Disse and protrude into the vascular lumen through endothelial fenestrations (Frevert et al., 1993, 1996; Cerami et al., 1994; Pradel et al., 2002; Coppi et al., 2007). The “gateway hypothesis,” which has predominated for several decades, suggests that sporozoites glide along the sinusoid wall until they find a Kupffer cell (Frevert et al., 2005), which they traverse to subsequently infect underlying hepatocytes. This hypothesis was supported by ultrastructural data suggesting that sporozoites specifically traverse Kupffer cells and not endothelial cells (Danforth et al., 1980; Meis et al., 1983; Vreden, 1994; Pradel et al., 2002). The molecular basis for this specific recognition is a key unresolved question of the early stages of Plasmodium development in its vertebrate host.We previously used a phage display library screening strategy to identify receptor–ligand combinations used by Plasmodium during its cycle in vector mosquitoes (Ghosh et al., 2001, 2009, 2011). Furthermore, blocking the interactions between parasite ligands and mosquito host cell receptors led to a significant reduction of malaria transmission by mosquitos (Ito et al., 2002). By screening a phage display library, we identified a peptide, P39, that binds to Kupffer cells and, by doing so, inhibits sporozoite entry. Further work determined that P39 interacts specifically with a major Kupffer cell surface protein, CD68, making this a candidate receptor for sporozoite traversal of Kupffer cells and liver infection.     

The Proteasome Inhibitor Epoxomicin Has Potent Plasmodium falciparum Gametocytocidal Activity

Malaria continues to be a major global health problem, but only a limited arsenal of effective drugs is available. None of the antimalarial compounds commonly used clinically kill mature gametocytes, which is the form of the parasite that is responsible for malaria transmission. The parasite that causes the most virulent human malaria, Plasmodium falciparum, has a 48-h asexual cycle, while complete sexual differentiation takes 10 to 12 days. Once mature, stage V gametocytes circulate in the peripheral blood and can be transmitted for more than a week. Consequently, if chemotherapy does not eliminate gametocytes, an individual continues to be infectious for several weeks after the clearance of asexual parasites. The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill all stages of intraerythrocytic parasites but do not affect the viability of human or mouse cell lines. Twenty-four hours after treatment with 100 nM epoxomicin, the total parasitemia decreased by 78%, asexual parasites decreased by 86%, and gametocytes decreased by 77%. Seventy-two hours after treatment, no viable parasites remained in the 100 or 10 nM treatment group. Epoxomicin also blocked oocyst production in the mosquito midgut. In contrast, the cysteine protease inhibitors epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester and N-acetyl-l-leucyl-l-leucyl-l-methioninal blocked hemoglobin digestion in early gametocytes but had no effect on later stages. Moreover, once the cysteine protease inhibitor was removed, sexual differentiation resumed. These findings provide strong support for the further development of proteasome inhibitors as antimalaria agents that are effective against asexual, sexual, and mosquito midgut stages of P. falciparum.The current recommended treatments for malaria caused by Plasmodium falciparum, including artemisinin combination therapy, eliminate intraerythrocytic asexual parasites that are responsible for the clinical symptoms. However, these treatments do not kill mature intraerythrocytic gametocytes that are required for the transmission of the parasite (24). In contrast to the 2-day asexual cycle of P. falciparum, the production of a mature stage V gametocyte takes 10 to 12 days. Once mature gametocytes are taken up by a mosquito during a blood meal, fertilization is stimulated. The resulting zygotes develop into oocysts where thousands of sporozoites are produced that can be transmitted to humans during a subsequent blood meal. The prolonged period required for P. falciparum gametocyte maturation in the human host suggests that malaria can be transmitted for several weeks after asexual parasites are eliminated (23). Thus, the development of drugs that are effective against both asexual-stage parasites and gametocytes may directly decrease malaria morbidity and mortality and reduce the spread of the disease.Cysteine protease and proteasome inhibitors have been found to affect asexual intraerythrocytic parasites and are being evaluated as possible antimalarial agents (4, 7, 10, 15, 19-21, 25). However, their effect on the 10- to 12-day course of intraerythrocytic gametocyte development has not been reported. Proteasome inhibitors also have not been tested on parasites taken up by a mosquito, while cysteine protease inhibitors have been shown to significantly decrease P. falciparum gamete surface antigen processing, oocyst production, and sporozoite maturation (7, 10). The dual cysteine and serine protease inhibitors l-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) also have been reported to reduce P. falciparum exflagellation and the transmission of Plasmodium berghei to mosquitoes (22, 26).Genes predicted to code for cysteine proteases and the proteasome are expressed throughout gametocytogenesis, providing targets for both classes of compounds (12, 28). Falcipain 1 and the P. berghei orthologs of PfSERA8 (PbECP1) and metacaspase 1 (PbMC1) are the only proteases whose function has been studied directly during gametocytogenesis by targeted gene disruption (3, 9, 14). The disruption of falcipain 1 and PfECP1 affected oocyst production in the mosquito midgut but not the asexual or sexual intraerythrocytic stage, while no stage of the life cycle was affected by the PbMC1 knockout. The work described here evaluates the effect of cysteine and proteasome inhibitors during P. falciparum sexual differentiation and development in the mosquito midgut.     

Piperaquine Pharmacodynamics and Parasite Viability in a Murine Malaria Model

Piperaquine (PQ) is an important partner drug in antimalarial combination treatments, but the long half-life of PQ raises concerns about drug resistance. Our aim was to investigate the extended antimalarial effect of PQ in a study of drug efficacy, reinoculation outcomes, and parasite viability after the administration of a single dose of PQ in the murine malaria model. Initially, male Swiss mice were inoculated with Plasmodium berghei and at 64 h after parasite inoculation were given PQ phosphate at 90 mg/kg of body weight intraperitoneally. Parasite viability, drug efficacy, reinoculation responses, and parasite resistance were determined at 25, 40, 60, 90, and 130 days after drug administration. At each time point, six mice were reinoculated with 10⁷ P. berghei parasites and blood was harvested from another four mice for viability passage into naïve mice (n = 5 for each blood sample) and from another two mice for determination of the plasma PQ concentration. The efficacy study demonstrated that the residual PQ concentrations did not suppress the infection after 25 days. Viable parasites were present up to 90 days after PQ dosing, although only 50% and 25% of the passaged parasites remained viable at 60 and 90 days postdosing, respectively. Viable parasites passaged into the naïve hosts were generally resistant to PQ when they were exposed to the drug for a second time. PQ was found to have a substantial antimalarial effect in this model, and the effect appears to be sufficient for a host immunological response to be established, resulting in the long-term survival of P. berghei-infected mice.Piperaquine (PQ) is a bisquinoline antimalarial drug used in contemporary artemisinin combination treatment (ACT) strategies as a partner to dihydroartemisinin (1, 4). In order to prevent drug resistance and early parasite recrudescence associated with the short-acting artemisinin drugs, ACT partner drugs should be long-acting schizonticides with half-lives (t_1/2s) exceeding 4 days, or two asexual parasite life cycles (10, 21). Recent pharmacokinetic studies have demonstrated that PQ has biphasic elimination and a long terminal t_1/2 of 12 to 28 days in humans (10, 15, 31, 32).Several clinical trials of the PQ-dihydroartemisinin combination have shown that it has a high degree of efficacy and good tolerability for the treatment of Plasmodium falciparum infections (1, 2, 5, 7, 11, 12, 17). While this combination is now considered the first-line antimalarial treatment in some Southeast Asian countries, the long t_1/2 of PQ raises concerns about adverse effects and drug resistance (6, 10, 20, 22). Detailed preclinical pharmacodynamic data for PQ, alone or in combination with artemisinin drugs, would complement clinical studies, especially when there is interest in the therapeutic impact of persistent, low PQ concentrations.We have recently demonstrated that PQ has a biphasic elimination profile in mice and has a terminal elimination t_1/2 in malaria parasite-infected and healthy mice of 18 days and 16 days, respectively (18). The pharmacodynamic component of our study revealed that after the administration of a single dose of PQ phosphate (10 to 90 mg/kg of body weight) there was a rapid reduction in the level of parasitemia to a subdetectable parasite density in the groups receiving high doses and recrudescence approximately 7 days later. In the group receiving the highest dose (90 mg/kg of body weight), a subclinical infection persisted for at least 60 days, at which time the plasma PQ concentration was estimated to be 20- to 100-fold lower than earlier therapeutic levels. However, reinoculation with P. berghei parasites did not cause the standard lethal infection that was found in control mice, suggesting that the mice treated with PQ had developed a degree of immunity to the parasites (18, 19). The present study was therefore conducted to investigate drug efficacy, reinoculation outcomes, and parasite viability after the administration of a single dose of PQ in the murine malaria model.     

Replication of Plasmodium in reticulocytes can occur without hemozoin formation,resulting in chloroquine resistance

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes.Clinical symptoms of malaria are associated with Plasmodium infection of RBCs. Human Plasmodium falciparum parasites catabolize more than half of the hemoglobin (Hb) in the RBCs (Goldberg, 2005). The amino acids derived from Hb proteolysis are used for protein synthesis and energy metabolism. The proteolysis of Hb is accompanied by the release of free heme, which is cytotoxic for the parasite and is rapidly detoxified and converted into hemozoin (Hz). Therefore, both Hb degradation and heme detoxification are considered to be essential for P. falciparum survival (Goldberg, 2005). The digestion of Hb is a conserved and semiordered process, which principally occurs within the acidified digestive vacuole (DV). After the initial cleavage by aspartic and papain-like cysteine endoproteases, Hb unfolds and becomes accessible to further proteolysis by downstream proteases. In the P. falciparum DV, there are four aspartic proteases (plasmepsins) and two papain-like cysteine proteases (falcipains) capable of hydrolyzing native Hb (Goldberg, 2005; Subramanian et al., 2009). Gene disruption studies of hemoglobinases demonstrated that P. falciparum has developed redundant and overlapping Hb degradation pathways, demonstrating the importance of Hb digestion for the parasite (Liu et al., 2006; Bonilla et al., 2007). However, Hb is a poor source of methionine, cysteine, glutamine, and glutamate; in addition, human Hb contains no isoleucine and P. falciparum blood-stage parasite growth is most effective in culture medium supplemented with these amino acids, especially isoleucine (Liu et al., 2006). These data indicate that P. falciparum parasites are not only dependent on Hb digestion, but also import exogenous amino acids (Liu et al., 2006; Elliott et al., 2008).Most studies on Hb degradation have been performed using P. falciparum maintained with mature RBCs (normocytes) in vitro. It is unknown whether observations on P. falciparum Hb digestion made in vitro can be directly translated to parasites replicating in vivo or for parasites developing in reticulocytes such as the human parasite P. vivax and P. ovale. For example, mechanisms of resistance to some drugs that interfere with Hb digestion and heme detoxification (e.g., chloroquine) differ between P. vivax and P. falciparum (Baird, 2004; Baird et al., 2012) indicating that there may be differences in their Hb digestion pathways.To obtain a better insight into Hb digestion in parasites developing in vivo we used a rodent malaria parasite, P. berghei, which preferentially invades reticulocytes. We show a high level of functional redundancy among the predicted hemoglobinases, as 6 of the 8 are dispensable in vivo. Unexpectedly, we were able to create parasite mutants lacking the enzymes known to initiate Hb digestion. These parasites were able to multiply in reticulocytes without Hz formation and were resistant to chloroquine.     

Assessment of Malaria In Vitro Drug Combination Screening and Mixed-Strain Infections Using the Malaria Sybr Green I-Based Fluorescence Assay

Several drug development strategies, including optimization of new antimalarial drug combinations, have been used to counter malaria drug resistance. We evaluated the malaria Sybr green I-based fluorescence (MSF) assay for its use in in vitro drug combination sensitivity assays. Drug combinations of previously published synergistic (atovaquone and proguanil), indifferent (chloroquine and azithromycin), and antagonistic (chloroquine and atovaquone) antimalarial drug interactions were tested against Plasmodium falciparum strains D6 and W2 using the MSF assay. Fifty percent inhibitory concentrations (IC₅₀s) were calculated for individual drugs and in fixed ratio combinations relative to their individual IC₅₀s. Subsequent isobologram analysis and fractional inhibitory concentration determinations demonstrated the expected drug interaction pattern for each combination tested. Furthermore, we explored the ability of the MSF assay to examine mixed parasite population dynamics, which are commonly seen in malaria patient isolates. Specifically, the capacity of the MSF assay to discern between single and mixed parasite populations was determined. To simulate mixed infections in vitro, fixed ratios of D6 and W2 strains were cocultured with antimalarial drugs and IC₅₀s were determined using the MSF assay. Dichotomous concentration curves indicated that the sensitive and resistant parasites composing the genetically heterogeneous population were detectable. Biphasic analysis was performed to obtain subpopulation IC₅₀s for comparison to those obtained for the individual malaria strains alone. In conclusion, the MSF assay allows for reliable antimalarial drug combination screening and provides an important method to discern between homogenous and heterogeneous parasite populations.Malaria is a severe global health problem that is compounded by the emergence of drug-resistant parasites. The emergence of these multidrug-resistant Plasmodium species, particularly P. falciparum, has made decisions regarding malaria chemoprophylaxis and treatment more complicated. Furthermore, it is predicted that increased incidences of clinical infections and subsequent deaths are likely as the rapid spread of resistant parasites occurs (15, 23, 35, 39). Several drug development strategies have been used to counter malaria drug resistance, including optimization of new antimalarial drug combinations. Several groups have examined drug combinations in laboratory settings that may have promising efficacies in clinical settings (5, 7, 30). Artemisinin-based combination therapies, one of the most successful therapeutic combinations, are currently used in areas where malaria is endemic (39). However, it is predicted that even with an aggressive prophylactic and treatment campaign, resistance to these drugs will certainly emerge (8). In fact, resistance to these combinations has already been observed. Wongsrichanalai and others reported the decreasing efficacy of the artesunate-mefloquine combination on the Cambodian-Thai border (41). Thus, there is a need to discover novel combinations between existing antimalarials and/or new chemical entities that can be used in the treatment of severe malaria.In addition to resistance to antimalarial combination therapies, there is also a concern with Plasmodium mixed infections. A mixed infection is defined as an infection with more than one type of species or genotype of Plasmodium (24). Although highly understudied, the implications of a mixed infection are profound. Mixed infections can cause a relapse as a result of emergence of the resistant subpopulation of parasites after the sensitive subpopulation has been eradicated by drug therapy. The existence of a resistant population may be a result of both divergent evolution, where parasites have acquired resistance mechanisms, and/or two cohabitating parasites when the individual is infected (16, 24). This phenomenon has been observed in areas of malaria endemicity in Africa and Southeast Asia, where the mixed-infection prevalence is as high as 30% (24). However, there has been conflicting evidence as to the true frequency of Plasmodium mixed infections (27, 34). Furthermore, this problem is confounded by the inability to properly identify and differentiate Plasmodium mixed infections.The [³H]hypoxanthine incorporation assay has been used as the gold standard in P. falciparum drug susceptibility testing (11). Despite the assay''s reliability and accuracy, it is very expensive, involves multiple processing steps, and requires special handling and waste disposal procedures. In contrast, dye-based technologies, such as those using 4′,6-diaminino-2-phenylindole, Pico green, YOYO-1, and Sybr green, have been shown to have comparable results to radioactive assays (2, 10, 17, 19, 36, 38). Many of these assays use DNA dye intercalation, which accurately measures parasite growth. Use of these assays has increased because they are relatively simple and inexpensive to run compared to their radioactive and enzyme-linked immunosorbent assay-based counterparts.While several in vitro drug sensitivity assays have been used to analyze antimalarial drug interactions, the ability of the malaria Sybr green I-based fluorescence (MSF) assay for this purpose has not been fully characterized. The [³H]hypoxanthine incorporation assay has been shown to detect differences in susceptibility patterns as well as being able to identify drug interactions (11, 30). However, radioactivity usage makes it costly and difficult to routinely use in research and clinical settings, particularly in a resource-limited environment. The MSF assay utilizes the Sybr green I dye (Invitrogen, San Diego, CA), which is relatively inexpensive and has been shown to reliably measure P. falciparum in vitro drug sensitivities. As the prevalence of Plasmodium mixed infections increases, there is a need for an assay that can reliably identify and differentiate drug-sensitive subpopulations in a particular infection. In this study, we examined the capability of the MSF assay to determine drug interactions and discern between single and mixed P. falciparum populations.     

Randomized,Double-Blind Study of the Safety,Tolerability, and Efficacy of Tafenoquine versus Mefloquine for Malaria Prophylaxis in Nonimmune Subjects

This study represents the first phase III trial of the safety, tolerability, and effectiveness of tafenoquine for malaria prophylaxis. In a randomized (3:1), double-blinded study, Australian soldiers received weekly malaria prophylaxis with 200 mg tafenoquine (492 subjects) or 250 mg mefloquine (162 subjects) for 6 months on a peacekeeping deployment to East Timor. After returning to Australia, tafenoquine-receiving subjects received a placebo and mefloquine-receiving subjects received 30 mg primaquine daily for 14 days. There were no clinically significant differences between hematological and biochemical parameters of the treatment groups. Treatment-related adverse events for the two groups were similar (tafenoquine, 13.4%; mefloquine, 11.7%). Three subjects on tafenoquine (0.6%) and none on mefloquine discontinued prophylaxis because of possible drug-related adverse events. No diagnoses of malaria occurred for either group during deployment, but 4 cases (0.9%) and 1 case (0.7%) of Plasmodium vivax infection occurred among the tafenoquine and mefloquine groups, respectively, up to 20 weeks after discontinuation of medication. In a subset of subjects recruited for detailed safety assessments, treatment-related mild vortex keratopathy was detected in 93% (69 of 74) of tafenoquine subjects but none of the 21 mefloquine subjects. The vortex keratopathy was not associated with any effect on visual acuity and was fully resolved in all subjects by 1 year. Tafenoquine appears to be safe and well tolerated as malaria prophylaxis. Although the volunteers'' precise exposure to malaria could not be proven in this study, tafenoquine appears to be a highly efficacious drug for malaria prophylaxis.The continuing spread of multidrug-resistant Plasmodium species and concerns about adverse effects associated with antimalarial drugs has made the prevention of malaria problematic for nonimmune subjects, such as tourists and soldiers who travel to malaria endemic areas. No antimalarial drug is completely effective in preventing malaria (10); however, an ideal prophylactic drug would be highly effective against all malaria-inducing species, very well tolerated, and taken infrequently to enhance compliance (21). Currently, mefloquine, doxycycline, and atovaquone-proguanil are recommended for malaria prophylaxis (5, 23). These drugs are highly effective in preventing malaria but have shortcomings that limit their effectiveness, such as adverse effects, expense, and the difficulty of monitoring daily compliance within deployed military populations. Furthermore, none of these recommended drugs prevents the development and relapse of Plasmodium vivax and P. ovale dormant liver stages (hypnozoites).Tafenoquine, a long-acting 8-aminoquinoline, is currently being codeveloped by GlaxoSmithKline (GSK) Research & Development Limited and the Walter Reed Army Institute of Research as a replacement for primaquine and for the prevention of malaria. Like primaquine, tafenoquine produces hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient recipients (21). Tafenoquine acts on all stages of the malaria parasite, with the potential to protect against all species of malaria parasites. Previous studies with a challenge model (4) and of indigenous populations in areas in which malaria is endemic have shown that tafenoquine was highly efficacious in preventing P. falciparum malaria and well tolerated (9, 13, 21). Tafenoquine was also shown to be efficacious in preventing both P. falciparum and P. vivax malaria for up to 6 months in Thai soldiers (22).This first phase III study of tafenoquine for malaria prophylaxis was a randomized, double-blind, active controlled study carried out with healthy Australian soldiers deployed to East Timor as part of a United Nations (UN) peacekeeping mission. The primary study objective was to compare the safety and tolerability of tafenoquine with those of mefloquine in malaria prophylaxis for 6 months. A subset of 98 subjects underwent extra safety assessments to investigate the possible effects of phospholipidosis, methemoglobin, and cardiac safety. Since a placebo arm to document exposure was not possible, the key secondary objective was to assess the efficacy of tafenoquine in preventing P. falciparum and P. vivax malaria during and following deployment.(This study was presented in part at the 51st Annual Meeting of the American Society of Tropical Medicine and Hygiene, Denver, CO, November 2002.)     

Imidocarb Dipropionate Clears Persistent Babesia caballi Infection with Elimination of Transmission Potential

Antimicrobial treatment of persistent infection to eliminate transmission risk represents a specific challenge requiring compelling evidence of complete pathogen clearance. The limited repertoire of antimicrobial agents targeted at protozoal parasites magnifies this challenge. Using Babesia caballi as both a model and a specific apicomplexan pathogen for which evidence of the elimination of transmission risk is required for international animal movement, we tested whether a high-dose regimen of imidocarb dipropionate cleared infection from persistently infected asymptomatic horses and/or eliminated transmission risk. Clearance with elimination of transmission risk was supported by the following four specific lines of evidence: (i) inability to detect parasites by quantitative PCR and nested PCR amplification, (ii) conversion from seropositive to seronegative status, (iii) inability to transmit infection by direct inoculation of blood into susceptible recipient horses, and (iv) inability to transmit infection by ticks acquisition fed on the treated horses and subsequently transmission fed on susceptible horses. In contrast, untreated horses remained infected and capable of transmitting B. caballi using the same criteria. These findings establish that imidocarb dipropionate treatment clears B. caballi infection with confirmation of lack of transmission risk either by direct blood transfer or a high tick burden. Importantly, the treated horses revert to seronegative status according to the international standard for serologic testing and would be permitted to move between countries where the pathogen is endemic and countries that are free of the pathogen.Antimicrobial therapy is primarily directed to reducing pathogen load below levels associated with disease, and treatment efficacy is most commonly evaluated by improvement in clinical signs (23, 27). Asymptomatic persistent infections represent an important subset of infections and present specific challenges for antimicrobial therapy (21, 24). The goal of therapy in persistent infections is clearance of the pathogen to prevent future relapse to clinical disease and/or transmission to additional susceptible hosts. Thus, achieving and confirming pathogen clearance become paramount in the treatment of persistent infections.The taxonomic range of pathogens that establish asymptomatic persistent infection is extremely broad, from RNA viruses to eukaryotic parasites (8, 15, 26). Among the latter, apicomplexan parasites in the genera Babesia, Plasmodium, and Theileria illustrate both the difficulty of effecting clearance with a limited repertoire of antimicrobial drugs and confirming that clearance and the elimination of subsequent transmission risk have been achieved (10, 21, 25, 29). These pathogens may persist in immunocompetent hosts at levels below the limits of routine microscopic detection and without overt signs of disease and yet serve as efficient reservoirs for arthropod vector-borne transmission (10, 19, 26, 28). Babesia caballi exemplifies this pattern: horses that recover from acute disease, when parasitemia levels exceed 10⁶ parasites per ml of blood, progress to an asymptomatic phase with parasitemia below 10⁵ parasites per ml of blood (18, 26). Acute B. caballi infection is characterized by high fever (>40°C), anemia, anorexia, malaise, tachypnea, and dyspnea (9). Following the acute phase, horses remain persistently infected and serve as reservoirs for transmission by tick vectors (26). Areas of endemicity for B. caballi include parts of Africa, the Middle East, Asia, Central and South America, the Caribbean, and Europe (9). While this hemoprotozoan parasite is widespread in tropical and subtropical regions, infecting horses, mules, donkeys, and zebras, many temperate-region countries are free of B. caballi infection and prohibit entry of infected horses (14). Consequently, the importation of horses into B. caballi-free countries or regions requires clearance of infection from persistently infected asymptomatic horses and confirmation of infection-free status. This requirement has a significant impact on the international movement of horses highly valued for either breeding or competition (5, 14). In this study, we tested whether imidocarb dipropionate eliminated B. caballi from persistently infected horses and, consequently, the risk of transmission by either direct blood transfer or tick vectors (the natural route of transmission). Furthermore, we tested if imidocarb dipropionate treatment resulted in reversion to seronegative status according to the international standard for importation of horses into infection-free countries.     

In Vitro Sensitivities of Plasmodium falciparum to Different Antimalarial Drugs in Uganda

The control of malaria is challenged by resistance of Plasmodium falciparum to multiple drugs. New combination regimens are now advocated for the treatment of uncomplicated falciparum malaria, but the extent of resistance to newer agents is incompletely understood. We measured the in vitro sensitivity of P. falciparum parasites cultured from children enrolled in a drug efficacy trial in Kampala, Uganda, from 2006 to 2008. Sensitivities were measured by comparing levels of histidine-rich protein-2 in parasites incubated with different concentrations of drugs with those in untreated controls. The cultured parasites exhibited a wide range of sensitivities to chloroquine (CQ); monodesethylamodiaquine (MDAQ), the major active metabolite of amodiaquine; and quinine (QN). Mean 50% inhibitory concentration (IC₅₀) results were above standard cutoffs for resistance for CQ and MDAQ. Parasites were generally sensitive to dihydroartemisinin (DHA), lumefantrine (LM), and piperaquine (PQ). For CQ, MDAQ, and QN but not the other drugs, activities against individual strains were highly correlated. We also assessed known resistance-mediating polymorphisms in two putative transporters, pfcrt and pfmdr1. When parasites that were least and most sensitive to each drug were compared, the pfmdr1 86Y mutation was significantly more common in parasites that were most resistant to CQ and MDAQ, and the pfmdr1 D1246Y mutation was significantly more common in parasites that were most resistant to MDAQ and QN. In summary, we demonstrated in parasites from Kampala a range of sensitivities to older drugs; correlation of sensitivities to CQ, MDAQ, and QN; and good activity against nearly all strains for DHA, LM, and PQ.Resistance of Plasmodium falciparum to available drugs remains a major challenge to the control of malaria. Older drugs, including the aminoquinolines chloroquine (CQ) and amodiaquine (AQ) and the antifolate sulfadoxine/pyrimethamine (SP), are already seriously compromised, with unacceptable levels of treatment failure in most of Africa (61). In the setting of increasing drug resistance, the WHO has recommended artemisinin-based combination therapy (ACT) for the treatment of uncomplicated falciparum malaria (42). The commonly used ACTs in Africa are artemether/lumefantrine (AM/LM), artesunate/amodiaquine (AS/AQ), and dihydroartemisinin/piperaquine (DHA/PQ), each containing an artemisinin combined with a longer-acting drug. These ACTs have shown excellent efficacy for the treatment of malaria in Africa. However, there is concern that heavy use of ACTs will offer strong selective pressure for parasites with diminished sensitivity to the drugs. This development may seriously jeopardize the efficacy of ACTs.Multiple recent studies in Africa have demonstrated excellent efficacy of AM/LM, AS/AQ, and DHA/PQ for the treatment of falciparum malaria (8, 18, 29, 30, 34, 46, 63, 64). Clinical trials provide our primary means of assessing antimalarial drug efficacy, but they offer only an indirect measure of the sensitivity of parasites to drugs because outcomes can be affected by multiple factors independent of drug sensitivity, including compliance with treatment regimens, drug absorption, pharmacokinetics, antimalarial immunity, and human genetic polymorphisms. In addition, clinical trials are not a sensitive means of identifying early selection of parasites with diminished drug sensitivity, as moderate decreases in sensitivity may have limited impact on clinical outcomes.The sensitivity of malaria parasites to drugs can be evaluated directly using parasites cultured in vitro. Systems for the culture of P. falciparum are well established, although the adaptation of parasites from an active infection to culture remains somewhat problematic. Thus, information on the in vitro drug sensitivity of cultured parasites is limited. Available studies have shown a wide range of sensitivities to older drugs. Parasites with diminished sensitivity to CQ, AQ, and SP are commonly seen (61). For newer drugs, including the active artemisinin metabolite DHA and the ACT partner drugs LM and PQ, African studies have suggested good sensitivity of most parasites (4, 6, 10, 48, 49). However, there is reason for concern that increasing use of ACTs and monotherapies may select for parasites with resistance to important newer agents. First, one study demonstrated parasites with markedly diminished sensitivity to artemether from French Guiana and Senegal (28). Second, recent data from Cambodia have shown diminished responsiveness to artesunate and prolonged parasite clearance times, suggesting the emergence of parasites with diminished sensitivity to artemisinins (15, 39, 54). Third, poor in vitro sensitivity to AQ and its active metabolite monodesethylamodiaquine (MDAQ) has already been demonstrated in Africa (55), parasite resistance-mediating polymorphisms predicted poor response to treatment with AQ and were selected by prior AQ therapy (12, 13, 23, 25, 27, 43), and use of an AQ-containing regimen selected for parasites with diminished in vitro drug sensitivity in subsequent new infections (36). Fourth, treatment with AM/LM selected in subsequent infections for parasites with polymorphisms that may lead to diminished drug responsiveness (14, 27, 31, 57). Fifth, PQ has a history of widespread resistance after broad use as monotherapy some decades ago in China (9). African studies have generally demonstrated good sensitivity of African parasites to piperaquine, but some parasites with in vitro 50% inhibitory concentrations (IC₅₀s) over 100 nM have been identified (6, 10, 35).In some cases, parasite genetic polymorphisms that mediate decreased drug responsiveness are known. The K76T polymorphism in the putative transporter pfcrt is the key mediator of resistance to CQ (11, 21) and also impacts response to AQ (13, 23). Mutations in another putative transporter, pfmdr1, appear to decrease sensitivity to CQ, AQ, and QN, and some of the same mutations may increase sensitivity to other drugs, including mefloquine and halofantrine, both of which are related to LM, and artemisinin. Increases in pfmdr1 copy number decrease in vitro sensitivity to mefloquine, halofantrine, LM, QN, and artemisinin (56) and have clearly been associated with mefloquine treatment failure (50). However, some pfmdr1 polymorphisms (S1034C and N1042D) and increases in pfmdr1 gene copy number are generally not seen in Africa (14, 25, 59), and our understanding of the importance of the pfmdr1 polymorphisms that are common in Africa (N86Y, Y184F, and D1246Y) is incomplete.In order to better characterize the sensitivity of P. falciparum from Kampala, Uganda, to relevant antimalarial drugs, we collected parasites causing uncomplicated malaria in a cohort of children enrolled in a drug efficacy trial, determined the sensitivity of these parasites to six key antimalarial drugs, and searched for associations between sensitivities to different drugs and between in vitro drug sensitivity and parasite genotypes. We found that parasites in Kampala exhibit a broad range of drug sensitivities, especially to CQ, MDAQ, and QN; that sensitivities to these three drugs, but not other tested drugs, were tightly correlated; and that polymorphisms in pfmdr1 were associated with but did not fully explain resistance to these drugs.     

Analysis of Single-Nucleotide Polymorphisms in the crt-o and mdr1 Genes of Plasmodium vivax among Chloroquine-Resistant Isolates from the Brazilian Amazon Region

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.Plasmodium vivax is the most widely distributed human malaria parasite, causing approximately 80 to 300 million clinical cases of malaria each year (17). Numerous factors indicate that this burden will increase due to the emergence and spread of chloroquine-resistant parasites (3, 17).More than 50% of the malaria cases in Latin America occur in Brazil, and P. vivax predominates as the causative agent (16, 21). Notably, failures of chloroquine treatment of P. vivax malaria in the Brazilian Amazon city of Manaus have been reported recently (1). The local confirmation of the presence of active P. vivax parasites resisting chloroquine at the proposed minimal effective concentration in plasma for sensitive strains is a public health concern deserving attention.Point mutations in two digestive-vacuole membrane proteins of P. falciparum, the P. falciparum chloroquine resistance transporter (PfCRT) and multidrug resistance 1 protein (PfMDR1), have been associated with chloroquine resistance (CQR), albeit to different extents (2, 10). Orthologues of these proteins in P. vivax (P. vivax CRT-O [PvCRT-O] and PvMDR1) have been identified previously (6, 15, 18), and recently, pvmdr1 mutant alleles were suggested to be associated with both in vitro and in vivo CQR in Southeast Asia (6, 20).Here, we report a single-nucleotide polymorphism (SNP) analysis of pvmdr1 and pvcrt-o genes in P. vivax isolates from chloroquine-treated patients with and without recrudescent disease in the Brazilian Amazon region. In addition to complete coding sequences, we analyzed sequences from 5′ flanking regions and introns.Field isolates were collected during a 28-day in vivo chloroquine efficacy study conducted in the city of Manaus, Brazil (8). Plasmatic chloroquine levels in all volunteers were measured by high-performance liquid chromatography on day 3 to confirm adequate dosing and good absorption of the oral chloroquine intake (three doses of 10, 7.5, and 7.5 mg/kg of body weight in 150-mg tablet form at 24-h intervals). Clinical treatment failure was defined as the occurrence of a positive blood smear result (confirmed by PCR diagnostic analysis) on day 14, 21, or 28 and the presence of parasites in peripheral blood (collected on the same day as the positive blood smear) containing >10 ng/ml of chloroquine as determined by high-performance liquid chromatography (7). Measurements of chloroquine and its active metabolite desethylchloroquine in whole blood were not obtained, as plasma samples were collected and processed at and transported from remote field sites. The presence of drug-resistant isolates in plasma samples with a mean chloroquine concentration ± standard deviation of 356.6 ± 296.1 ng/ml, however, undoubtedly confirms CQR (4). Using these stringent criteria, we selected seven samples (four obtained prior to treatment [on day 0] from patients with nonrecrudescent disease and three obtained after treatment [on days 21 and 28] from other patients with recrudescent disease). Different sets of primers, PCR conditions, and algorithms were used to amplify coding and noncoding regions and to generate and analyze sequences (see the supplemental material).Analyses of the complete coding sequences of the pvmdr1 gene demonstrated that these sequences contained 24 SNPs and a single conserved microsatellite sequence. Notably, 17 (73%) of the 24 SNPs detected were nonsynonymous; 11 were contained in predicted extracellular loops in the parasite digestive-vacuole cytosol, and 5 were present in transmembrane domains (TMDs) (Fig. ​(Fig.1;1; also see Table S2 in the supplemental material). Despite the high frequency of SNPs, however, none were found in ABC conserved motifs (see Table S3 in the supplemental material).Open in a separate windowFIG. 1.Predicted structures of and representative polymorphisms in PvMDR1 and PfMDR1. PvMDR1, like PfMDR1, has two hydrophobic homologous domains, each with six transmembrane α-helices, and a cytosolic domain harboring nucleotide-binding domain 1 (NBD₁) and NBD₂, each containing an ATP-binding site with characteristic Walker motifs A and B and the S signature (ATP-binding cassette) of these transporters. In this figure, boxes with dotted lines in the diagram of PvMDR1 delimit predicted NBD locations. In the PfMDR1 illustration, closed dots represent point mutations associated with CQR in P. falciparum. In the PvMDR1 illustration, open dots represent SNPs described previously (5, 6, 11, 18, 20), open triangles represent SNPs identified in this study, and patterned dots represent SNPs described in other studies (5, 6, 11, 18, 20).To relate these polymorphisms to protein function, we mapped selective constraints throughout coding sequences by calculating a K_d/K_s (ω) ratio for each amino acid substitution (see Fig. S1 in the supplemental material). These results revealed ω values of ≥1, indicating higher accelerated rates of nonsynonymous substitutions than expected to result from chance at the sites of most SNPs (P < 0.00001). Indeed, an ω value of 1.80811 (P < 0.00001), evidencing positive selection, was calculated for nucleotide positions 2722 to 2727, which correspond to amino acids 907 and 908 in PvMDR1. Presently, however, it is difficult to ensure that this positive selection is advantageous for the CQR phenotype and not for another metabolic aspect(s) related to the function of the PvCRT-O and PvMDR1 transporters. Like other authors (5, 6, 11, 18), we did not find SNPs at homologous positions of PfMDR1. We did, however, find a polymorphism in PvMDR1 at amino acid position 89, which is located very near and in the same intravacuolar loop, between TMDs I and II, as an SNP at the corresponding position in PfMDR1 (amino acid position 86), which has partial correlation with the CQR phenotype (12, 20). In addition, SNP Y976F, proposed previously as an early marker of CQR (6, 20), was found in only one isolate from a patient with nonrecrudescent disease. It is thus clear that the value of these polymorphisms as markers of CQR in P. vivax needs to be further explored.Complete sequencing of the 14 exons of pvcrt-o revealed the presence of one synonymous transition and five nonsynonymous substitutions (Fig. ​(Fig.2;2; see also Table S4 in the supplemental material). Interestingly, the lysine (K) insertion at amino acid 10 of PvCRT-O, highly prevalent in Thai isolates (20), was also found in a sample from a chloroquine-treated patient from Brazil with recrudescent disease. Moreover, the rate of nonsynonymous substitutions in TMD VII was significantly higher than expected to result from chance (P < 0.05) (see Fig. S2 in the supplemental material). Despite these differences, however, we were unable to find an association between chloroquine treatment failure and the SNPs detected. Similar results following SNP analyses of pvcrt-o in monkey-adapted strains and human isolates have been reported previously (5, 15, 20).Open in a separate windowFIG. 2.Predicted structures of and representative polymorphisms in PvCRT-O and PfCRT. Like PfCRT, PvCRT-O has 10 predicted transmembrane helices, with C- and N-terminal domains located in the parasite cytoplasm. In the diagram of PvCRT-O, open dots represent SNPs detected in chloroquine-resistant and chloroquine-sensitive samples by Nomura et al. (15), and open triangles represent (named) SNPs described in this work. Closed dots in the PfCRT illustration represent point mutations that have been strongly associated with CQR in P. falciparum.The lack of mutations in coding sequences of pvmdr1 and pvcrt-o associated with treatment failure prompted us to look for these associations in introns and 5′ flanking sequences, as they influence gene expression in malaria (7, 13). Interestingly, 6 (46%) of 13 introns contained SNPs, nine of which were transitions and one of which was a transversion (see Table S5 in the supplemental material). The remaining introns contained conserved microsatellites (see Table S6 in the supplemental material). Yet differences in length at the microsatellite in intron 12 (see Fig. S3 in the supplemental material) among samples sharing the same evolutionary spatial and temporal distributions suggest that this microsatellite marker may be a good candidate for the study of this locus. Moreover, analyses of 5′ flanking sequences from pvmdr1 and pvcrt-o revealed a single SNP (A→G) at nucleotide position 566 upstream from the ATG start codon in pvcrt-o. This high degree of conservation is consistent with the results of another study which analyzed untranslated regions of pfmdr1 in six reference strains with different chloroquine phenotypes (14). Together, these results suggest the existence of functional constraints at these genome loci that may play an important role in gene regulation.In conclusion, we have analyzed the complete coding and noncoding sequences of the pvmdr1 and pvcrt-o genes from Brazilian P. vivax isolates that fulfilled rigorous criteria for chloroquine sensitivity and resistance. Although we did not genotype these isolates to determine if CQR isolates represent clonal as opposed to complex populations, it is clear that CQR isolates are already circulating in the Brazilian Amazon basin. Therefore, these sequences can be used as a baseline for future prospective studies of drug resistance in this region. Our analysis revealed, however, that there was no correlation between CQR and pvmdr1 and pvcrt-o mutations in these specific isolates, even if some mutations had been not randomly selected. The striking differences in the topologies of SNPs in the MDR1 and CRT transporter genes between P. vivax and P. falciparum thus indicate that mechanisms other than mutations may be implicated in the appearance of CQR in these two human malaria parasites (likely candidates are gene amplification and changes in expression levels [9, 11, 19]) or that other genes are associated with this virulent phenotype in P. vivax.     

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).     

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.     

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.