Reversal of chloroquine resistance in falciparum malaria

Control of falciparum malaria infections has been increasingly hampered by the emergence of parasites resistant to chloroquine, pyrimethomine and other standard anti-malarials. Chloroquine-resistant strains of Plasmodium falciparum, for example, which originally appeared in South-East Asia and South America are now found in East Asia and sub-Saharan Africa(1). Attempts to combat this alarming development have to date taken two main forms: (1) the judicious use of existing ontimalarials, preferably in combinations, in an attempt to delay the emergence of resistance; and (2) on aggressive research effort aimed at identifying a new generation of antimalarial drugs. But what i f it became possible to administer an antimalarial drug together with a second drug capable of overcoming resistance to the first? A recent report from Samuel Martin and co-workers at The Walter Reed Army Institute of Research in Washington DC raises just such an intriguing possibility.     

Impact of chloroquine resistance on malaria mortality

Over 12 years, from 1984 to 1995, we conducted a prospective study of overall and malaria specific mortality among three rural populations in the Sahel, savanna and forest areas of Senegal. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality in each of the studied populations. After the emergence of chloroquine resistance, the risk of malaria death among children 0–9 years old in the three populations was multiplied by 2.1, 2.5 and 5.5, respectively. This is the first study to document malaria mortality at the community level in Africa before and after the emergence of chloroquine resistance. Findings suggest that the spread of chloroquine resistance has had a dramatic impact on the level of malaria mortality in most epidemiological contexts in tropical Africa.     

Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism

Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations.     

Did medicated salt hasten the spread of chloroquine resistance in Plasmodium falciparum?

With acknowledged difficulties in achieving satisfactory compliance rates for the large-scale delivery of many antiporositic drugs, the use of medicated salt has often been seen as a useful way to improve the level o f treatment in target populations. Iodinated salt is said to have contributed to a decline in endemic goitre, and salt medicated with chloroquine and/or other antimalorials, or with diethylcarbomazine, has been widely used in public health programmes against malaria and filariosis respectively. In this article, however, David Payne suggests that chloroquinized salt programmes may have been a major factor in promoting chloroquine resistance in Plasmodium falciparum.     

The current status of drug resistance in malaria

Drug resistant malaria is a major health problem; it poses a threat to the lives of millions of people and renders it less possible for the worldwide eradication programme to attain its goal in the foreseeable future. At present Plasmodium falciparum is resistant to varying degrees to all antimalarial drugs available e.g. chloroquine, sulfadoxine and pyrimethamine, quinine and even to the new compound, mefloquine.Chloroquine-resistant P. falciparum originated in Thailand some 25 years ago has spread in all directions to Southeast Asia, Western Pacific, to central and southeast India, East Africa and West Africa. In South America, it started in Colombia and now affects the whole of Central and South America with the exception of Argentina, Paraguay and Peru which practically have no falciparum malaria.The mechanism of drug resistance in malaria parasites is believed to be due to gene mutation selected under drug pressure. It may be one-step as in pyrimethamine or multi-step as in chloroquine. Resistant mutation occurs both in schizogony and sporogony. The parasites lose their S strains through hybridization or overgrowth, shifting in character progressively towards high grade resistance.Policies that may help to minimise further development of resistance to existing compounds and to safeguard any new drugs that may be developed in the future include (1) limit the distribution of antimalarials; (2) select priority groups for prophylaxis; (3) use the gametocidal drug primaquine to restrict transmission of resistant strains; (4) establish an effective drug monitoring system; (5) only deploy drugs for control as part of an integrated campaign; (6) control use of new antimalarial; (7) encourage the use of tested effective drug regimens for treatment and (8) encourage research on antimalarials.     

Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the world's burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.     

Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.     

Chemotherapy and drug resistance in malaria

Over recent years many antimalarial drugs have been rendered useless by the development of resistance by the malaria parasite. New antimalarials are rapidly suffering the same fate as the traditional therapies and yet a biological understanding of the mechanisms of resistance has, until recently, not been described. This review describes recent work which has identified the mechanism of resistance to the dihydrofolate reductase (DHFR) inhibitors as being due to point mutations within the DHFR gene that render the enzyme less susceptible to inhibition by the drugs. The relationship between chloroquine resistance and the recently described multidrug resistance gene is explored and the possibility that this is the main cause of chloroquine resistance by the parasite is discussed. Parasites have developed resistance against many of the quinine-like antimalarials over the past three decades and the possibility that this is linked to the appearance of chloroquine resistance must be considered.     

Reduction of malaria transmission to Anopheles mosquitoes with a six-dose regimen of co-artemether

BackgroundResistance of malaria parasites to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) is increasing in prevalence in Africa. Combination therapy can both improve treatment and provide important public health benefits if it curbs the spread of parasites harbouring resistance genes. Thus, drug combinations must be identified which minimise gametocyte emergence in treated cases, and so prevent selective transmission of parasites resistant to any of the partner drugs.ConclusionsCo-artemether is highly effective at preventing post-treatment transmission of P. falciparum. Our results suggest that co-artemether has specific activity against immature sequestered gametocytes, and has the capacity to minimise transmission of drug-resistant parasites.     

Evolutionary paradigm of chloroquine-resistant malaria in India

Drug pressure in the field is believed to be responsible for the emergence of drug-resistant Plasmodium falciparum, the parasite that causes malaria. Variants of the P. falciparum chloroquine resistance transporter (pfcrt) gene have been shown to be responsible for conferring resistance to the commonly used drug chloroquine. In particular, an amino acid mutation, K76T, was shown to have a strong positive correlation with the chloroquine-resistant varieties of malaria parasites. Global studies have reported highly reduced genetic diversity surrounding K76T in the pfcrt gene, which indicates that the mutation has been a target of positive Darwinian natural selection. However, two recent studies of P. falciparum in India found high genetic diversity in the pfcrt gene, which, at first sight, do not support the role of natural selection in the evolution of chloroquine resistance in India.     

HIV and malaria: a lesson in immunology?

HIV is now common in many areas of Africa that are also highly endemic for malaria. In this article, Geoff Butcher summarizes the available data on the possible interaction o f HIV and malaria, and shows that the course of falciparum malaria is virtually unaffected by the presence of HIV. This raises significant questions for our understanding of immunity to the asexual blood stages of human malaria and the use of animal models in malaria research.     

Efflux of a range of antimalarial drugs and ‘chloroquine resistance reversers’ from the digestive vacuole in malaria parasites with mutant PfCRT

Chloroquine‐resistant malaria parasites (Plasmodium falciparum) show an increased leak of H⁺ ions from their internal digestive vacuole in the presence of chloroquine. This phenomenon has been attributed to the transport of chloroquine, together with H⁺, out of the digestive vacuole (and hence away from its site of action) via a mutant form of the parasite's chloroquine resistance transporter (PfCRT). Here, using transfectant parasite lines, we show that a range of other antimalarial drugs, as well as various ‘chloroquine resistance reversers’ induce an increased leak of H⁺ from the digestive vacuole of parasites expressing mutant PfCRT, consistent with these compounds being substrates for mutant forms, but not the wild‐type form, of PfCRT. For some compounds there were significant differences observed between parasites having the African/Asian Dd2 form of PfCRT and those with the South American 7G8 form of PfCRT, consistent with there being differences in the transport properties of the two mutant proteins. The finding that chloroquine resistance reversers are substrates for mutant PfCRT has implications for the mechanism of action of this class of compound.     

Quantitative trait loci controlling refractoriness to Plasmodium falciparum in natural Anopheles gambiae mosquitoes from a malaria-endemic region in western Kenya

Natural anopheline populations exhibit much variation in ability to support malaria parasite development, but the genetic mechanisms underlying this variation are not clear. Previous studies in Mali, West Africa, identified two quantitative trait loci (QTL) in Anopheles gambiae mosquitoes that confer refractoriness (failure of oocyst development in mosquito midguts) to natural Plasmodium falciparum parasites. We hypothesize that new QTL may be involved in mosquito refractoriness to malaria parasites and that the frequency of natural refractoriness genotypes may be higher in the basin region of Lake Victoria, East Africa, where malaria transmission intensity and parasite genetic diversity are among the highest in the world. Using field-derived F2 isofemale families and microsatellite marker genotyping, two loci significantly affecting oocyst density were identified: one on chromosome 2 between markers AG2H135 and AG2H603 and the second on chromosome 3 near marker AG3H93. The first locus was detected in three of the five isofemale families studied and colocalized to the same region as Pen3 and pfin1 described in other studies. The second locus was detected in two of the five isofemale families, and it appears to be a new QTL. QTL on chromosome 2 showed significant additive effects while those on chromosome 3 exhibited significant dominant effects. Identification of P. falciparum-refractoriness QTL in natural An. gambiae mosquitoes is critical to the identification of the genes involved in malaria parasite transmission in nature and for understanding the coevolution between malaria parasites and mosquito vectors.     

South American Plasmodium falciparum after the malaria eradication era: clonal population expansion and survival of the fittest hybrids

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999-2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006-2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.     

Plasmodium falciparum: in vitro drug interaction between chloroquine and enantiomers of amlodipine

Both enantiomers of amlodipine, whose calcium antagonist action resides almost exclusively in the R(-) enantiomer, reversed chloroquine resistance in Plasmodium falciparum in vitro. R(-) enantiomer was slightly more effective than the S(+) enantiomer in potentiating chloroquine action against chloroquine-resistant strains of parasites. No potentiating effect was observed in chloroquine-sensitive parasites. Both enantiomers entered rapidly into parasitized erythrocytes to the same extent. Reversal of chloroquine resistance by the enantiomers of amlodipine was related to dose-dependent increase in the accumulation of chloroquine inside the erythrocytes parasitized by resistant parasites. These results suggest that the potentiating effect on chloroquine is independent of calcium metabolism of malaria parasites.     

PfCRT-mediated drug transport in malarial parasites

A wide range of drug transport studies using intact infected red blood cells, isolated malarial parasites, heterologous expression systems, and purified protein, combined with elegant genetic experiments, have suggested that chloroquine transport by the Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key aspect of the molecular mechanism of quinoline antimalarial drug resistance. However, many questions remain. This short review summarizes data that have led to drug channel versus drug pump hypotheses for PfCRT and suggests ways in which recent contrasting interpretations might be reconciled.     

Diversity of Plasmodium falciparum chloroquine resistance transporter (pfcrt) exon 2 haplotypes in the Pacific from 1959 to 1979

Nearly one million deaths are attributed to malaria every year. Recent reports of multi-drug treatment failure of falciparum malaria underscore the need to understand the molecular basis of drug resistance. Multiple mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) are involved in chloroquine resistance, but the evolution of complex haplotypes is not yet well understood. Using over 4,500 archival human serum specimens collected from 19 Pacific populations between 1959 and 1979, the period including and just prior to the appearance of chloroquine treatment failure in the Pacific, we PCR-amplified and sequenced a portion of the pfcrt exon 2 from 771 P. falciparum-infected individuals to explore the spatial and temporal variation in falciparum malaria prevalence and the evolution of chloroquine resistance. In the Pacific, the prevalence of P. falciparum varied considerably across ecological zones. On the island of New Guinea, the decreases in prevalence of P. falciparum in coastal, high-transmission areas over time were contrasted by the increase in prevalence during the same period in the highlands, where transmission was intermittent. We found 78 unique pfcrt haplotypes consisting of 34 amino acid substitutions and 28 synonymous mutations. More importantly, two pfcrt mutations (N75D and K76T) implicated in chloroquine resistance were present in parasites from New Hebrides (now Vanuatu) eight years before the first report of treatment failure. Our results also revealed unexpectedly high levels of genetic diversity in pfcrt exon 2 prior to the historical chloroquine resistance selective sweep, particularly in areas where disease burden was relatively low. In the Pacific, parasite genetic isolation, as well as host acquired immune status and genetic resistance to malaria, were important contributors to the evolution of chloroquine resistance in P. falciparum.     

Gazing at a giraffe gyroscope: where are we going?

Giraffe are popular animals to watch while on wildlife safaris, and feature prominently in zoos, advertisements, toys and cartoons. Yet, until recently, few field studies have focused on giraffe. We introduce this giraffe topic issue with a review essay that explores five primary questions: How many (sub) species of giraffe exist? What are the dynamics of giraffe herds? How do giraffe communicate? What is the role of sexual selection in giraffe reproduction? How many giraffe reside in Africa? A confluence of causes has produced drastic declines in giraffe population numbers in Africa, and we conclude that guiding giraffe conservation plans depends upon evaluation of the five key quandaries that we pose.     

The efficacy of antifolate antimalarial combinations in Africa: a predictive model based on pharmacodynamic and pharmacokinetic analyses

At present, effective treatment for non-severe malaria is the most important malaria control strategy in Africa. Pyrimethamine-sulfadoxine (PSD) is rapidly becoming the first-line treatment in areas of chloroquine resistance, although the parasite chemoresistance factors that dispose towards clinical failure with PSD are still unclear. Here, Bill Watkins and colleagues analyse the relationship between the pharmacokinetic properties of two treatment combinations (PSD and chlorproguanil-dapsone) in vivo and the respective in vitro isobolograms for parasites with specific drug-resistance patterns. From this relationship, they develop a hypothesis that may explain clinical drug failure and differential efficacy between treatments. The deductions can be tested in field studies to validate or refute the model.