the mechanisms of resistance to antimalarial drugs in plasmodium falciparum

O R I G I N A L

A R T I C L E

The mechanisms of resistance toantimalarial drugs in Plasmodium falciparum

Jacques Le Bras*, Remy DurandLaboratory of Parasitology, University of Paris V and Bichat-Claude Bernard Hospital, 75018 Paris, France

I N T R O D U C T I O N

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, accom-

panied by an increase in malaria-related mortality [2].

Keywords

drugs,

genetics,

Plasmodium,

resistance

Received 2 July 2002;

revised 16 October 2002;

accepted 27 January 2003

*Correspondence and reprints:

[email protected]

paris.fr

A B S T R A C T

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 chemo-

prophylaxis 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 sulphadoxine–pyrimeth-

amine 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, mefloquine and halofantrine) are still unclear. Epidemio-

logical 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 multidrug-

resistance 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.

� 2003 Blackwell Publishing Fundamental & Clinical Pharmacology 17 (2003) 147–153 147

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.

M E C H A N I S M O F C H L O R O Q U I N E

R E S I S T A N C E

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 deficits were because of changes in

the pH gradient or to altered membrane permeability

related to an efflux mechanism, or both. Chloroquine-

resistance is reversible by verapamil, which modulates

resistance in the cancer cells of multi-drug-resistant

(MDR) mammals. This discovery led to the identification

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 amplifica-

tion of this gene with chloroquine-resistance. Chloro-

quine transport is altered in the cells with modified 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 specificity; this suggests the involvement of

either a specific 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 chloroquine-

resistant phenotype [8]. Transfection of this genotype

suffices to confer chloroquine resistance. Four independ-

ent mutation profiles are seen, varying geographically:

Asia–Africa, Papua, South America 1 and 2. The major

event in chloroquine resistance thus is the emergence in

Indo–China 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. Nonethe-

less, the wild isolates, which are very polymorphous,

have not been totally replaced; they still represent

approximately half the strains in circulation.

R E S I S T A N C E T O P Y R I M E T H A M I N E ,

P R O G U A N I L A N D O T H E R

A N T I M E T A B O L I T E S

Because Plasmodium in humans can capture and use the

host’s 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 cyclo-

guanil, the active metabolite in proguanil (Paludrine�;

AstraZeneca, Macclesfield, 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 asso-

ciated 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

148 J. Le Bras and R. Durand

� 2003 Blackwell Publishing Fundamental & Clinical Pharmacology 17 (2003) 147–153

in the recombinant dihydrofolate reductase (DHFR) of

the parasite reduces the drug’s affinity without affecting

the enzyme’s operation on its natural substrate. Multiple

mutations diminish the efficacy 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 define the resistance

threshold as an IC50 of 50 nmol/L of cycloguanil. For

PfDHFR, 95% of 148 isolates from travellers with a wild-

type 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 physiologi-

cal conditions, the blood folate concentration can

influence 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 sulfadoxine-

resistant parents demonstrate the close association

between the PfDHPS mutations and resistance and a

complete correlation between multiple PfDHFR muta-

tions 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 ¼ 3/3) or LTF (n ¼ 6/17), and all those

(n ¼ 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 ¼ 11), RI (n ¼ 8), RII

(n ¼ 4) and RIII (n ¼ 1); all those (n ¼ 21) with three or

more mutations of PfDHFR had grades RI–RIII 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�; GlaxoSmith-

Kline; Uxbridge, UK) to treat strains resistant to sulfa-

doxine/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 mal-

aria. As with the antifolates, use of atovaquone alone

against P. falciparum leads to rapid selection for resistant

mutants [21].

M U L T I P L E - D R U G C H E M O R E S I S T A N C E

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

Figure 1 Metabolicpathway of pyrimi-

dines in Plasmodium falciparum and

targets of antifolates (DHPS and DHFR)

and atovaquone (cytochrome b).

Chemoresistance of Plasmodium falciparum 149


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 pyrimethamine–sulfa-

doxine (Fansidar�) resistance, to the extent to the

latter is combined with or used to back up chloroq-

uine. 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 phenom-

enon linked to the common or shared aspects of their

modes of action and probably of their resistance mech-

anisms. A close correlation was observed between

sensitivity to cycloguanil and pyrimethamine in 314 iso-

lates from Africa (r ¼ 0.9, personal data). The parasites

Figure 2 Map of malaria risk in Africa.



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 mefloquine (Lariam� [Roche] and Mepha-

quin� [Mepha]) and halofantrine (Halfan� [GlaxoSmith-

Kline]). A correlation analysis is the first 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 lyso-

somotropic antimalarials in a population of parasites.

Accordingly, in the African P. falciparum strains impor-

ted into France, the variation of the level of sensitivity to

monodesethyl–amodiaquine, the active metabolite of

amodiaquine, can be explained by the level of chloroq-

uine sensitivity. Amodiaquine may thus fail in subjects

infested by highly chloroquine-resistant strains. A sim-

ilar rationale may account for halofantrine failures with

mefloquine-resistant strains. The inverse correlation

observed between chloroquine and mefloquine or halo-

fantrine reflects an opposite trend: chloroquine-sensitive

strains are substantially less sensitive to mefloquine 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 resist-

ance, as do mefloquine and halofantrine [24]. Clinical

evidence however does not confirm frequent cross-

resistance 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 anti-

malarials or independent selection of resistance to each

compound (Table II). Important and multiple drug pres-

sure has existed since the beginning of the 1950s, with

the combined use of chloroquine, amodiaquine, quinine,

and then after 1980, mefloquine as lysosomotropic

agents. Multiple resistance has not been observed in

the absence of substantial utilization of the correspond-

ing 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

efficacy of antimalarials is highly heterogeneous, as

numerous authors have observed, with excellent res-

ponse to doses less than the standard chloroquine or

quinine regimen (in the absence of immunity) or poor

Table I Countries classified by order of

increased frequency of Plasmodium falci-

parum 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.

Chloroquine-R Cycloguanil-RPercentage with dual resistancea

% (n) % (n) Theoreticalb Observed 95% CI

Madagascar 0 (6) 15 (20) 0 0 0–46

Mauritania 25 (4) 23 (13) 5.8 5.8 0–66

Mali 38 (47) 18 (157) 6.8 6.7 1.4–18

Burkina Faso 25 (12) 32 (34) 8.1 11 0.6–42

Cote d’Ivoire 34 (113) 38 (324) 13 12 6.4–20

Central African Republic 28 (18) 51 (49) 14 12 22–36

Senegal 31 (67) 46 (171) 14 17 8.9–28

(FRI) Comoros 40 (25) 41 (93) 16 21 4.6–49

Guinea 50 (10) 54 (39) 27 24 4.3–60

Ghana 40 (5) 69 (16) 27 29 2.1–78

D.R. Congo 42 (12) 81 (47) 34 31 8.8–63

Benin 57 (14) 77 (66) 44 35 9.4–70

Congo 56 (16) 80 (60) 45 44 20–70

Kenya 68 (9) 63 (16) 42 48 16–82

Cameroon 61 (67) 80 (183) 49 50 36–64

Togo 57 (7) 89 (36) 51 52 15–88

Gabon 93 (14) 67 (39) 62 66 31–91

Totalc 43 (472) 51 (1489) 22 22 19–26

Cycloguanil-R: PfDHFR S108N; Chloroquine-R: CI50 > 100 nmol/L.aDual resistance to both drugs.bProduct of the frequency of resistance to each component.cImported, including from countries not listed.

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



response to standard mefloquine doses (before its intro-

duction into these regions). The latter case nonetheless

did not involve a situation of multiple drug resistance.

C O N C L U S I O N

The absence of rigorous clinical studies on the chemo-

resistance of P. falciparum (with blood assays for

antimalarials) make it difficult to determine the respect-

ive 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. Amplification of genes linked

to chemoresistance has not been observed but overex-

pression of proteins or differences in drug accumulation

could explain the differences in sensitivity for the same

mutation profiles. We currently lack information about

the back-mutation of resistance and the possible disad-

vantages of mutant strains relative to the wild type.

Regionally, the levels and proportions of chemoresis-

tance are highly heterogeneous and can probably be

explained by drug pressure, in particular, from slow-

elimination drugs such as sulfadoxine–pyrimethamine or

mefloquine. A single point mutation, easily selected by

drug pressure, is sufficient 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 efficacy of proguanil or pyrimethamine, given alone.

The proportion of isolates with three or more mutations,

resistant to the sulfadoxine/pyrimethamine combina-

tion, is not known. Chloroquine resistance, which is

more complex, probably corresponds to several simulta-

neous but rare mutations (that probably appeared alone

at the end of the 1950s and then dispersed by migra-

tion). It involves a phenotype that prevents accumula-

tion 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 fluctuations.

Resistance to quinine, mefloquine and halofantrine is

probably because of mechanisms of the type observed for

chloroquine. Unlike chloroquine, however, these com-

pounds 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 fortu-

nately 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 anti-

malarials 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.

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falciparum.

Resistant mutations (additional mutations)

Antimalarial Causal Associated

Chloroquine Pfcrt K76T (A220S) Pfmdr1 N86Y,

Pfcg2 j14

Amodiaquine – –

Quinine – –

Mefloquine – Pfmdr1 N86Y

Halofantrine – Pfmdr1 N86Y

Lumefantrine – –

Cycloguanil Pfdhfr S108N + N51I ou C59R,

Pfdhfr S108T + A16V

–

Pyrimethamine Pfdhfr S108N (N51I, C59R, I164L) –

Sulfadoxine Pfdhps A437G (K540E) –

Atovaquone – Pfcytb Y268N

Artemether – –



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the mechanisms of resistance to antimalarial drugs in plasmodium falciparum

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