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.     

Intensity of transmission and spread of gene mutations linked to chloroquine and sulphadoxine-pyrimethamine resistance in falciparum malaria

The number of malaria parasite clones per infection-multiplicity of parasite clones-is affected by the transmission intensity, multiplicity increases with increasing transmission. This affects the frequency of parasites' sexual recombination and, if several mutations in different genes are involved, can break down drug resistant genotypes. Therefore, the effects of malaria transmission intensity on the spread of drug resistance could vary depending on the number of genes involved. Here we show that, compared to low transmission, intermediate-high transmission is associated with a 20-100-fold lower risk for the mutations linked to chloroquine resistance and a 6-17 times higher risk for those linked to sulphadoxine-pyrimethamine resistance. This is consistent with the hypothesis of a multigenic basis for chloroquine resistance and a monogenic basis for that of sulphadoxine-pyrimethamine. Reducing transmission intensity could slow the spread of resistance. However, a reduction below a critical threshold (e.g. when parasite prevalence in children 2-9 years old is around 60-80%) could, paradoxically, accelerate the spread of resistance to chloroquine and possibly to other drug combinations whose basis is multigenic. Our findings have important implications for malaria control because increasing drug resistance has a substantial impact on mortality.     

The development and spread of drug-resistant malaria

Resistance of Plasmodium falciparum to chloroquine has emerged in the late 1950s and has now conquered the large majority of areas where this species is endemic. Resistance to alternative drugs has already occurred in several parts of the world and has become a particularly serious problem in Thailand. Emergence and spread of resistance are the result of interactions between parasite, humans, vector and drugs, enhanced by particular ecological features. The control of malaria transmission by means other than drugs would probably curb the propagation of resistance but current health care policies offer only limited prospects for the reactivation or implementation of systematic malaria control before the potential of the affordable antimalarials has been exhausted. In this article, Walther Wernsdorfer considers the epidemiological factors associated with the development and spread of drug-resistant malaria.     

Using evolutionary costs to enhance the efficacy of malaria control via genetically manipulated mosquitoes

An earlier mathematical model exploring the use of genetically manipulated mosquitoes for malaria control suggested that the prevalence of malaria is reduced significantly only if almost all mosquitoes become completely resistant to malaria. Central to the model was the 'cost of resistance': the reduction of a resistant mosquito's evolutionary fitness in comparison with a sensitive one's. Here, we consider the possibility of obtaining more optimistic outcomes by taking into account the epidemiological (in addition to the evolutionary) consequences of a cost of resistance that decreases the life-span of adult mosquitoes (the most relevant parameter for the parasite's epidemiology). There are two main results. First, if despite its cost, resistance is fixed in the population, increasing the cost of resistance decreases the intensity of transmission. However, this epidemiological effect is weak if resistance is effective enough to be considered relevant for control. Second, if the cost of resistance prevents its fixation, increasing it intensifies transmission. Thus, the epidemiological effect of the cost of resistance cannot compensate for the lower frequency of resistant mosquitoes in the population. Overall, our conclusion remains pessimistic: so that genetic manipulation can become a promising method of malaria control, we need techniques that enable almost all mosquitoes to be almost completely resistant to infection.     

Epidemiological evidence for an association between chloroquine resistance of Plasmodium falciparum and its immunological properties

The appearance of chloroquine-resistant genotypes o f Plasmodium falciparum has thwarted the goal of global eradication of malaria. Although much effort has been put into understanding the molecular mechanisms of chloroquine resistance, many questions about its distribution remain open: Why, some 30 years after the emergence o f chloroquine resistance, have resistant genotypes not taken over the population? Why have many parasites remained sensitive? Why, after its first appearance in Africa, has chloroquine resistance spread so rapidly through sub-Saharan Africa? In this paper Jacob Koella reviews epidemiological data that suggest that an answer to these questions may involve an association between chloroquine resistance and immunological properties o f malaria parasites.     

A model for the coevolution of immunity and immune evasion in vector-borne diseases with implications for the epidemiology of malaria

We describe a model of host-parasite coevolution, where the interaction depends on the investments by the host in its immune response and by the parasite in its ability to suppress (or evade) its host's immune response. We base our model on the interaction between malaria parasites and their mosquito hosts and thus describe the epidemiological dynamics with the Macdonald-Ross equation of malaria epidemiology. The qualitative predictions of the model are most sensitive to the cost of the immune response and to the intensity of transmission. If transmission is weak or the cost of immunity is low, the system evolves to a coevolutionarily stable equilibrium at intermediate levels of investment (and, generally, at a low frequency of resistance). At a higher cost of immunity and as transmission intensifies, the system is not evolutionarily stable but rather cycles around intermediate levels of investment. At more intense transmission, neither host nor parasite invests any resources in dominating its partner so that no resistance is observed in the population. These results may help to explain the lack of encapsulated malaria parasites generally observed in natural populations of mosquito vectors, despite strong selection pressure for resistance in areas of very intense transmission.     

Fitness costs of resistance to antimalarial drugs

It has been recently reported that the prevalence of mutations associated with chloroquine resistance declined during the dry season. Fitness costs of drug resistance were suggested to be responsible for reduced survival of mutant parasites, and only parasites surviving chronic infections were transmitted at the onset of the rainy season. This implies that during seasonal transmission, significant changes can occur in allele frequency over the course of months, rather than years. The practical consequences of these findings for monitoring dynamics of drug-resistance markers are: (i) in areas of seasonal transmission, the sampling date matters; (ii) fluctuations in mutation frequencies might be explained by seasonality; and (iii) a much-awaited experimental determination of fitness costs of drug resistance becomes within reach.     

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.     

Effect of transmission reduction by insecticide-treated bednets (ITNs) on antimalarial drug resistance in western Kenya

Despite the clear public health benefit of insecticide-treated bednets (ITNs), the impact of malaria transmission-reduction by vector control on the spread of drug resistance is not well understood. In the present study, the effect of sustained transmission reduction by ITNs on the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) in children under the age of five years was investigated during an ITN trial in Asembo area, western Kenya. During the ITN trial, the national first line antimalarial treatment changed from CQ to SP. Smear-positive samples collected from cross sectional surveys prior to ITN introduction (baseline, n = 250) and five years post-ITN intervention (year 5 survey, n = 242) were genotyped for single nucleotide polymorphisms (SNPs) at dhfr-51, 59, 108, 164 and dhps-437, 540 (SP resistance), and pfcrt-76 and pfmdr1-86 (CQ resistance). The association between the drug resistance mutations and epidemiological variables was evaluated. There were significant increases in the prevalence of SP dhps mutations and the dhfr/dhps quintuple mutant, and a significant reduction in the proportion of mixed infections detected at dhfr-51, 59 and dhps-437, 540 SNPs from baseline to the year 5 survey. There was no change in the high prevalence of pfcrt-76 and pfmdr1-86 mutations. Multivariable regression analysis further showed that current antifolate use and year of survey were significantly associated with more SP drug resistance mutations. These results suggest that increased antifolate drug use due to drug policy change likely led to the high prevalence of SP mutations 5 years post-ITN intervention and reduced transmission had no apparent effect on the existing high prevalence of CQ mutations. There is no evidence from the current study that sustained transmission reduction by ITNs reduces the prevalence of genes associated with malaria drug resistance.     

Application of a multi-faceted approach for the assessment of treatment response in falciparum malaria: a study from Malaysian Borneo

Thirty-two patients reporting to the Lundu District Hospital, Sarawak, Malaysian Borneo, with uncomplicated falciparum malaria were recruited into a multifaceted study to assess treatment response. Following combined chloroquine and sulphadoxine/pyrimethamine treatment the patients were followed for 28 days according to the World Health Organisation in vivo drug response protocol. The in vivo study revealed that 13 (41%) of the patients had a sensitive response to treatment, five (16%) cleared asexual stage parasites but had persistent gametocytes, 11 (34%) had RI type resistance and three (9%) had RII type resistance requiring quinine intervention before day 7 for parasite clearance. Although clinically insignificant, patients with persistent gametocytes, surviving chloroquine and sulphadoxine/pyrimethamine treatment during maturation, were placed in the reduced response to treatment group for analysis. Allelic typing detected 100% prevalence of the pfcrt K76T marker associated with chloroquine resistance and 78% prevalence of the pfdhfr NRNL haplotype associated with sulphadoxine/pyrimethamine treatment failure. High serum chloroquine levels and pfdhfr haplotypes with 相似文献   

Evidence for activation of endogenous transporters in Xenopus laevis oocytes expressing the Plasmodium falciparum chloroquine resistance transporter, PfCRT

A large body of genetic, reverse genetic, and epidemiological data has linked chloroquine-resistant malaria to polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum. To investigate the biological function of the chloroquine resistance transporter, PfCRT, as well as its role in chloroquine resistance, we functionally expressed this protein in Xenopus laevis oocytes. Our data show that PfCRT-expressing oocytes exhibit a depolarized resting membrane potential and a higher intracellular pH compared with control oocytes. Pharmacological and electrophysiological studies link the higher intracellular pH to an enhanced amiloride-sensitive H(+) extrusion and the low membrane potential to an activated nonselective cation conductance. The finding that both properties are independent of each other, together with the fact that they are endogenously present in X. laevis oocytes, supports a model in which PfCRT activates transport systems. Our data suggest that PfCRT plays a role as a direct or indirect activator or modulator of other transporters.     

Plasmodium falciparum: differential selection of drug resistance alleles in contiguous urban and peri-urban areas of Brazzaville, Republic of Congo

The African continent is currently experiencing rapid population growth, with rising urbanization increasing the percentage of the population living in large towns and cities. We studied the impact of the degree of urbanization on the population genetics of Plasmodium falciparum in urban and peri-urban areas in and around the city of Brazzaville, Republic of Congo. This field setting, which incorporates local health centers situated in areas of varying urbanization, is of interest as it allows the characterization of malaria parasites from areas where the human, parasite, and mosquito populations are shared, but where differences in the degree of urbanization (leading to dramatic differences in transmission intensity) cause the pattern of malaria transmission to differ greatly. We have investigated how these differences in transmission intensity affect parasite genetic diversity, including the amount of genetic polymorphism in each area, the degree of linkage disequilibrium within the populations, and the prevalence and frequency of drug resistance markers. To determine parasite population structure, heterozygosity and linkage disequilibrium, we typed eight microsatellite markers and performed haplotype analysis of the msp1 gene by PCR. Mutations known to be associated with resistance to the antimalarial drugs chloroquine and pyrimethamine were determined by sequencing the relevant portions of the crt and dhfr genes, respectively. We found that parasite genetic diversity was comparable between the two sites, with high levels of polymorphism being maintained in both areas despite dramatic differences in transmission intensity. Crucially, we found that the frequencies of genetic markers of drug resistance against pyrimethamine and chloroquine differed significantly between the sites, indicative of differing selection pressures in the two areas.     

Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents

Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of crossresistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance. This was accompanied by restoration of efficient chloroquine binding to hematin in these selected lines. This S163R mutation provides insight into a mechanism by which PfCRT could gate the transport of protonated chloroquine through the digestive vacuole membrane. Evidence for the presence of this mutation in a Southeast Asian isolate supports the argument for a broad role for PfCRT in determining levels of susceptibility to structurally diverse antimalarials.     

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.     

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.     

Carriers, channels and chloroquine efficacy in Guinea-Bissau

Recent studies suggest that chloroquine resistance is mediated by an energy-dependent saturable chloroquine efflux carrier. An alternative explanation is that resistance is mediated by a channel. In Guinea-Bissau high doses of chloroquine are effective, well-tolerated and commonly used. This suggests that chloroquine resistance can be overcome by higher doses. Research on the mechanism of chloroquine resistance is of utmost importance and should include the effect of higher doses.     

The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily

The malaria parasite's chloroquine resistance transporter (CRT) is an integral membrane protein localized to the parasite's acidic digestive vacuole. The function of CRT is not known and the protein was originally described as a transporter simply because it possesses 10 transmembrane domains. In wild-type (chloroquine-sensitive) parasites, chloroquine accumulates to high concentrations within the digestive vacuole and it is through interactions in this compartment that it exerts its antimalarial effect. Mutations in CRT can cause a decreased intravacuolar concentration of chloroquine and thereby confer chloroquine resistance. However, the mechanism by which they do so is not understood. In this paper we present the results of a detailed bioinformatic analysis that reveals that CRT is a member of a previously undefined family of proteins, falling within the drug/metabolite transporter superfamily. Comparisons between CRT and other members of the superfamily provide insight into the possible role of the protein and into the significance of the mutations associated with the chloroquine resistance phenotype. The protein is predicted to function as a dimer and to be oriented with its termini in the parasite cytosol. The key chloroquine-resistance-conferring mutation (K76T) is localized in a region of the protein implicated in substrate selectivity. The mutation is predicted to alter the selectivity of the protein such that it is able to transport the cationic (protonated) form of chloroquine down its steep concentration gradient, out of the acidic vacuole, and therefore away from its site of action.     

Evidence for a pfcrt-associated chloroquine efflux system in the human malarial parasite Plasmodium falciparum

Resistance to the antimalarial drug chloroquine has been linked with polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum, yet the mechanism by which this gene confers the reduced drug accumulation phenotype associated with resistance is largely unknown. To investigate the role of pfcrt in mediating chloroquine resistance, we challenged P. falciparum clones differing only in their pfcrt allelic form with the "varying-trans" procedure. In this procedure, movement of labeled substrate across a membrane is measured when unlabeled substrate is present on the trans side of the membrane. If a transporter is mediating the substrate flow, a stimulation of cis-to-trans movement may be observed with increasing concentrations of trans substrate. We present evidence for an association of those pfcrt alleles found in chloroquine-resistant P. falciparum strains with the phenomenon of stimulated chloroquine accumulation under varying-trans conditions. Such an association is not seen with polymorphisms within pfmdr1, which encodes a homologue of the human multidrug resistance efflux pump. Our data are interpreted in terms of a model in which pfcrt is directly or indirectly involved in carrier-mediated chloroquine efflux from resistant cells.     

Plasmodium chabaudi: in vivo effects of Ca2+ antagonists on chloroquine-resistant and chloroquine-sensitive parasites

The effects of Ca2+ antagonists, verapamil, nicardipine, and diltiazem, on susceptibility to chloroquine were examined in mice infected with chloroquine-sensitive and chloroquine-resistant lines of Plasmodium chabaudi. In mice that received no chloroquine, daily injections of 50 mg/kg of verapamil, nicardipine, or diltiazem did not affect the growth of both sensitive and resistant parasites. When mice were injected daily with verapamil plus 2 to 3 mg/kg chloroquine, the chloroquine-sensitive parasite became more susceptible to chloroquine than the parasite in mice given chloroquine alone. On the other hand, in mice infected with chloroquine-resistant parasites, verapamil severely suppressed the growth of the parasite when accompanied by daily injections of 2 to 3 mg/kg of chloroquine, at which doses resistant parasites grew steadily in the absence of verapamil, indicating reversal of chloroquine resistance. This reversal was dose-dependent between 5 and 50 mg/kg of verapamil. Daily injections of nicardipine or diltiazem at 50 mg/kg also reversed resistance to chloroquine in resistant parasites. These results indicate that Ca2+ antagonists increase the susceptibility to chloroquine in a sensitive line of P. chabaudi and reverse chloroquine resistance in a resistant line.     

Plasmodium falciparum transmission rate and selection for drug resistance: a vexed association or a key to successful control?

While malaria eradication campaigns once adopted a combination of vector control and chemotherapy to overcome the disease, today's opinion on the matter is equivocal. So what has changed? This paper reviews some of the confusing hypotheses on the relationship between Plasmodium falciparum transmission and levels of drug resistance. New field evidence showing variations of in vivo chloroquine resistance in relation to indoor residual spraying and natural endemicity patterns, is considered with a view to how these phenomena implicate on control.