ORIGINAL ARTICLE

The mechanisms of resistance to antimalarial drugs in Plasmodium falciparum

my Durand Jacques Le Bras*, Re
Laboratory of Parasitology, University of Paris V and Bichat-Claude Bernard Hospital, 75018 Paris, France

Keywords
drugs, genetics, Plasmodium, resistance

ABSTRACT

Received 2 July 2002; revised 16 October 2002; accepted 27 January 2003

*Correspondence and reprints: jacques.lebras@bch.ap-hopparis.fr

Drug-resistant malaria is primarily caused by Plasmodium falciparum, a species highly prevalent in tropical Africa, the Amazon region and South-east Asia. It causes severe fever or anaemia that leads to more than a million deaths each year. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality among inhabitants of some endemic regions. The rationale for chemoprophylaxis is weakening as multiple-drug resistance develops against well-tolerated drugs. Plasmodium falciparum drug-resistant malaria originates from chromosome mutations. Analysis by molecular, genetic and biochemical approaches has shown that (i) impaired chloroquine uptake by the parasite vacuole is a common characteristic of resistant strains, and this phenotype is correlated with mutations of the Pfmdr1, Pfcg2 and Pfcrt genes; (ii) one to four point mutations of dihydrofolate reductase (DHFR), the enzyme target of antifolates (pyrimethamine and proguanil) produce a moderate to high level of resistance to these drugs; (iii) the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (DHPS), their enzyme target; (iv) treatment with sulphadoxinepyrimethamine selects for DHFR variants Ile(51), Arg(59), and Asn(108) and for DHPS variants Ser(436), Gly(437), and Glu(540); (v) clones that were resistant to some traditional antimalarial agents acquire resistance to new ones at a high frequency (accelerated resistance to multiple drugs, ARMD). The mechanisms of resistance for amino-alcohols (quinine, meoquine and halofantrine) are still unclear. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among isolated parasite populations, while resistance to antifolates is highly prevalent in most malarial endemic countries. Established and strong drug pressure combined with low antiparasitic immunity probably explains the multidrugresistance encountered in the forests of South-east Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilize at the same level as chloroquine-sensitive malaria. Nevertheless, resistance levels may differ according to place and time. In vivo and in vitro tests do not provide an adequate accurate map of resistance. Biochemical tools at a low cost are urgently needed for prospective monitoring of resistance.

INTRODUCTION Plasmodium falciparum, which is responsible for fatal malaria in humans, is a major cause of morbidity and mortality throughout the tropics. Each year in Africa,

approximately 500 million cases of falciparum malaria occur, and nearly 2 million people die [1]. This major public health problem is aggravated by the widespread diffusion of chloroquine resistance in P. falciparum, accompanied by an increase in malaria-related mortality [2].

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Chemoresistance, which concerns mainly falciparum, the parasite for the most serious type of malaria, is one of the factors in the worldwide upsurge of malaria in tropical regions. The chemoresistance of P. vivax involves the antimetabolites and for the past several years in Asia, chloroquine. Chloroquine remains generally effective for P. vivax, P. ovale and P. malariae. The resistance phenotype is determined by culturing the parasite. It is not necessarily expressed by treatment failure (particularly in subjects who have acquired defenses during previous malaria episodes); inversely, chemoresistance is only one possible cause of treatment failure. The mechanisms by which the parasite is chemoresistant to antimalarials generally involve chromosomal mutations. Parasite genetics, which has now been studied for a decade, should illuminate the epidemiology of chemoresistance. Essentially, malaria involves the interaction of three populations (humans, anopheles and Plasmodium), and the parasite adapts through mitotic mutations and meiotic recombinations. These parasites are extremely diverse, and a subject is infected on average by three different strains (from the bite of one or several mosquitoes) [3]. This diversity is assumed to be based on a high variation in allele frequency in the initial phase of mutant selection by drug pressure, a balance of selection and mutation that varies according to population size, with low levels of mixing in the islands, and high levels in dense African populations. MECHANISM OF CHLOROQUINE RESISTANCE The most spectacular characteristic of chloroquine is its capacity to concentrate itself from nanomolar levels outside the parasite to millimolar levels in the digestive vacuole of the intraerythrocytic trophozoite. It is here that it inhibits haemoglobin degradation and forms complexes with haematin. The resistant isolates have in common an alteration in the chloroquine accumulation in the digestive vacuole. Previous theories suggested that these accumulation decits were because of changes in the pH gradient or to altered membrane permeability related to an efux mechanism, or both. Chloroquineresistance is reversible by verapamil, which modulates resistance in the cancer cells of multi-drug-resistant (MDR) mammals. This discovery led to the identication of the protein Pgh1 in the digestive vacuole membrane of P. falciparum; it is an analog to the glycoproteins overexpressed in cancer cells where they function as pumps expelling cytotoxic drugs (ATP-binding cassette transporters). Resistance was initially attributed to

variations in the number of copies of the corresponding gene, the pfMDR1 gene, perhaps combined with isolated mutations. There is, however, no evidence of amplication of this gene with chloroquine-resistance. Chloroquine transport is altered in the cells with modied Pgh1. The associations between isolated mutations of PfMDR1 and chloroquine remain uncertain [4]. It now appears that chloroquine resistance is related to diminished uptake of the drug. Chloroquine accumulation has high structural specicity; this suggests the involvement of either a specic transporter/permease or a molecule associated with hematin in the digestive vacuole [5]. The PfCRT gene, located on chromosome 7, codes for a transmembrane protein located in the digestive vacuole membrane [6]. A set of mutations of this gene is found in all natural isolates from clinical chloroquine treatment failures [7] and in vitro in isolates with a chloroquineresistant phenotype [8]. Transfection of this genotype sufces to confer chloroquine resistance. Four independent mutation proles are seen, varying geographically: AsiaAfrica, Papua, South America 1 and 2. The major event in chloroquine resistance thus is the emergence in IndoChina at the end of the 1950s of mutants selected by drug pressure; these have spread to 90% of the territory of P. falciparum in the 40 years since. Nonetheless, the wild isolates, which are very polymorphous, have not been totally replaced; they still represent approximately half the strains in circulation. RESISTANCE TO PYRIMETHAMINE, PROGUANIL AND OTHER ANTIMETABOLITES Because Plasmodium in humans can capture and use the hosts purines but not their pyrimidines, the parasites must synthesize the latter (Figure 1). Isolated mutations of the PfDHFR gene are the molecular bases of P. falciparum resistance to pyrimethamine and to cycloguanil, the active metabolite in proguanil (Paludrine; AstraZeneca, Maccleseld, UK) [9]. In vitro isolates of P. falciparum from failures of proguanil prophylaxis or sulfadoxine/pyrimethamine treatment present resistance simultaneously to cycloguanil and to pyrimethamine. The S108N substitution is the principal mutation associated with resistance to pyrimethamine or cycloguanil in Africa and in South-east Asia. Substitution S108T is also found in South America [9]. The most frequent additive mutations are N51I and C59R. The mean IC50 of cycloguanil increases with the number of mutations [10]. The simple substitution of Asn or Thr at codon 108

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Figure 1 Metabolicpathway of pyrimidines in Plasmodium falciparum and targets of antifolates (DHPS and DHFR) and atovaquone (cytochrome b).

in the recombinant dihydrofolate reductase (DHFR) of the parasite reduces the drugs afnity without affecting the enzymes operation on its natural substrate. Multiple mutations diminish the efcacy of the enzyme on dihydrofolate and thus suggest that additional mutations are unfavourable to the parasites in the absence of drug pressure [11]. The reports below dene the resistance threshold as an IC50 of 50 nmol/L of cycloguanil. For PfDHFR, 95% of 148 isolates from travellers with a wildtype codon in position 108 (Ser) were susceptible and 91% of 68 isolates with serine replaced by asparagine at codon 108 (S108N) were resistant [12,13]. The S108N mutation is selected in isolates of travellers taking proguanil prophylaxis [14]. In Cameroon, the isolates with an S108N mutation for PfDHFR, alone or combined with a mutation of codon 59, showed adequate in vivo clinical response to Fansidar (Roche, Switzerland), and those with three PfDHFR mutations responded with early or late treatment failure (LTF) [15]. In physiological conditions, the blood folate concentration can inuence the effect of sulfadoxine, which may explain the Fansidar failures on P. falciparum isolates with only an S108N mutation of PfDHFR. It was initially thought that the mutations on the dihydropteroate synthetase gene (PfDHPS, target of some antifolates) might be responsible for sulfadoxine resistance. That is, PfDHFR and PfDHPS mutants are selected during sulfadoxine/ pyrimethamine treatment [16]. The progeny of a genetic cross between sulfadoxine-sensitive and sulfadoxineresistant parents demonstrate the close association between the PfDHPS mutations and resistance and a complete correlation between multiple PfDHFR mutations and resistance [17]. The predictive value of the

DHPS mutations in the Wang study was poor. In Kenya and Tanzania, the isolates with a S108N mutation of PfDHFR, alone or combined with a codon 59 mutation, responded in vivo to Fansidar by an adequate clinical response (n 3/3) or LTF (n 6/17), and all those (n 13) with three PfDHFR mutations responded by either early or LTF [16]. The predictive value of the DHPS mutations in the Curtis study was also poor. In the Peruvian Amazon region, 24 isolates with single S108N or S108T mutations of PfDHFR showed the following in vivo responses to Fansidar: S (n 11), RI (n 8), RII (n 4) and RIII (n 1); all those (n 21) with three or more mutations of PfDHFR had grades RIRIII responses [18,19]. The predictive value of the DHPS mutations in this study was 21of 34. Cross-resistance has been demonstrated between cycloguanil and pyrimethamine, and the potential use of other antifolates such as chlorproguanil plus dapsone (LapDap; GlaxoSmithKline; Uxbridge, UK) to treat strains resistant to sulfadoxine/pyrimethamine seems limited [16]. Atovaquone, whose target is supposed to be cytochrome b in the pyrimidine metabolic pathway [20], can be used with proguanil (Malarone; GlaxoSmithKline) to treat malaria. As with the antifolates, use of atovaquone alone against P. falciparum leads to rapid selection for resistant mutants [21]. MULTIPLE-DRUG CHEMORESISTANCE Multiple-drug chemoresistance for malaria refers to the resistance to several antimalarial drugs that has been observed in P. falciparum. It can be simultaneous or cross-resistance. Simultaneous resistance results

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Figure 2 Map of malaria risk in Africa.

principally from the large-scale and simultaneous use of several antimalarials, which causes strong selective pressure. Accordingly, cycloguanil resistance is added to chloroquine resistance, as is pyrimethaminesulfadoxine (Fansidar) resistance, to the extent to the latter is combined with or used to back up chloroquine. The heterogeneity of these multiple-resistant strains corresponds to the distribution of the level of

malaria transmission (Figure 2) and drug pressure (Table I). Cross-resistance between antimalarials is a phenomenon linked to the common or shared aspects of their modes of action and probably of their resistance mechanisms. A close correlation was observed between sensitivity to cycloguanil and pyrimethamine in 314 isolates from Africa (r 0.9, personal data). The parasites

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Table I Countries classied by order of increased frequency of Plasmodium falciparum isolates resistant to chloroquine and cycloguanil, in the absence of chemoprophylaxis, in malaria imported from Africa and the Indian Ocean into France between 1996 and 2001.

Percentage with dual resistancea Chloroquine-R % ( n) Madagascar Mauritania Mali Burkina Faso te dIvoire Co Central African Republic Senegal (FRI) Comoros Guinea Ghana D.R. Congo Benin Congo Kenya Cameroon Togo Gabon Totalc 0 (6) 25 (4) 38 (47) 25 (12) 34 (113) 28 (18) 31 (67) 40 (25) 50 (10) 40 (5) 42 (12) 57 (14) 56 (16) 68 (9) 61 (67) 57 (7) 93 (14) 43 (472) Cycloguanil-R % (n) 15 (20) 23 (13) 18 (157) 32 (34) 38 (324) 51 (49) 46 (171) 41 (93) 54 (39) 69 (16) 81 (47) 77 (66) 80 (60) 63 (16) 80 (183) 89 (36) 67 (39) 51 (1489) Theoreticalb 0 5.8 6.8 8.1 13 14 14 16 27 27 34 44 45 42 49 51 62 22 Observed 0 5.8 6.7 11 12 12 17 21 24 29 31 35 44 48 50 52 66 22 95% CI 046 066 1.418 0.642 6.420 2236 8.928 4.649 4.360 2.178 8.863 9.470 2070 1682 3664 1588 3191 1926

Cycloguanil-R: PfDHFR S108N; Chloroquine-R: CI50 > 100 nmol/L.

a

Dual resistance to both drugs. Product of the frequency of resistance to each component.

b c

Imported, including from countries not listed.

The threshold of 50% of dual resistance was chosen to classify a country in zone III [26].

that have a high level of chloroquine resistance, as in South-east Asia, are generally resistant to amodiaquine as well (Flavoquine [Aventis, France] and Camoquin [Parke Davis, USA]). The same is probably true in this region for meoquine (Lariam [Roche] and Mephaquin [Mepha]) and halofantrine (Halfan [GlaxoSmithKline]). A correlation analysis is the rst indication of a common mode of action and perhaps of a common mechanism of resistance. It is possible to assess in vitro the response of a single strain to several antimalarials and therefore to compare the action of different lysosomotropic antimalarials in a population of parasites. Accordingly, in the African P. falciparum strains imported into France, the variation of the level of sensitivity to monodesethylamodiaquine, the active metabolite of amodiaquine, can be explained by the level of chloroquine sensitivity. Amodiaquine may thus fail in subjects infested by highly chloroquine-resistant strains. A similar rationale may account for halofantrine failures with meoquine-resistant strains. The inverse correlation observed between chloroquine and meoquine or halofantrine reects an opposite trend: chloroquine-sensitive strains are substantially less sensitive to meoquine or halofantrine and vice versa in Africa [22,23]. In vitro evidence thus tends to support the view that chloroquine

and amodiaquine share common mechanism of resistance, as do meoquine and halofantrine [24]. Clinical evidence however does not conrm frequent crossresistance between chloroquine and amodiaquine [25]. Similarly, epidemiologic evidence about the origin of multiple drug resistance observed in South-east Asia, in particular, does not allow us to determine whether we see a common mechanism of resistance for diverse antimalarials or independent selection of resistance to each compound (Table II). Important and multiple drug pressure has existed since the beginning of the 1950s, with the combined use of chloroquine, amodiaquine, quinine, and then after 1980, meoquine as lysosomotropic agents. Multiple resistance has not been observed in the absence of substantial utilization of the corresponding antimalarial. A single mechanism of resistance preventing the accumulation of several compounds by some P. falciparum strains in the border areas of Thailand may nonetheless explain the frequent multiple drug resistance in these regions (accelerated resistance to multiple drugs). In the grassland regions of Africa, the efcacy of antimalarials is highly heterogeneous, as numerous authors have observed, with excellent response to doses less than the standard chloroquine or quinine regimen (in the absence of immunity) or poor

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Table II Antimalarial-resistant mutations identied in Plasmodium falciparum.

Resistant mutations (additional mutations) Antimalarial Chloroquine Amodiaquine Quinine Meoquine Halofantrine Lumefantrine Cycloguanil Pyrimethamine Sulfadoxine Atovaquone Artemether Causal Pfcrt K76T (A220S) Pfdhfr S108N + N51I ou C59R, Pfdhfr S108T + A16V Pfdhfr S108N (N51I, C59R, I164L) Pfdhps A437G (K540E) Pfcytb Y268N Associated Pfmdr1 N86Y, Pfcg2 j14 Pfmdr1 N86Y Pfmdr1 N86Y

response to standard meoquine doses (before its introduction into these regions). The latter case nonetheless did not involve a situation of multiple drug resistance. CONCLUSION The absence of rigorous clinical studies on the chemoresistance of P. falciparum (with blood assays for antimalarials) make it difcult to determine the respective roles of clinical resistance on the one hand and dosage errors and defective compliance, very frequent in chemical prophylaxis and in treatment, on the other [26]. The types of resistance observed in the laboratory in P. falciparum are linked to one or several associated chromosomal mutations. Amplication of genes linked to chemoresistance has not been observed but overexpression of proteins or differences in drug accumulation could explain the differences in sensitivity for the same mutation proles. We currently lack information about the back-mutation of resistance and the possible disadvantages of mutant strains relative to the wild type. Regionally, the levels and proportions of chemoresistance are highly heterogeneous and can probably be explained by drug pressure, in particular, from slowelimination drugs such as sulfadoxinepyrimethamine or meoquine. A single point mutation, easily selected by drug pressure, is sufcient to create resistance to an antimetabolite. It appears in multiple foci, with a high frequency for PfDHFR. Half of the African isolates studied carry at least two mutations, enough to impede the efcacy of proguanil or pyrimethamine, given alone.

The proportion of isolates with three or more mutations, resistant to the sulfadoxine/pyrimethamine combination, is not known. Chloroquine resistance, which is more complex, probably corresponds to several simultaneous but rare mutations (that probably appeared alone at the end of the 1950s and then dispersed by migration). It involves a phenotype that prevents accumulation of the drug. Its overall prevalence in Africa is now around 50%, with no evidence of a major increase in the last decade, but with important geographic uctuations. Resistance to quinine, meoquine and halofantrine is probably because of mechanisms of the type observed for chloroquine. Unlike chloroquine, however, these compounds do not currently face high levels of resistance, and they thus remain, depending on the region, completely or partly active. The most critical situation is in the jungle areas of Thailand, Cambodia and Myanmar, where multiple drug chemoresistance has been frequent since 1980. This situation, which fortunately corresponds to a very low proportion of malaria worldwide, is a worrisome singularity that is nonetheless extremely useful for the study of multiple drug resistance and its treatments. As the alternating use of antimalarials in pandemic areas is illusory, it seems essential to combine them for treatment and for prophylaxis in order to limit the extension of resistant genotypes. REFERENCES
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