For personal use. Only reproduce with permission from The Lancet Publishing Group.

Since the first reports of chloroquine-resistant falciparum

malaria in southeast Asia and South America almost half a
century ago, drug-resistant malaria has posed a major
problem in malaria control. By the late 1980s, resistance to
sulfadoxine-pyrimethamine and to mefloquine was also
prevalent on the Thai-Cambodian and Thai-Myanmar (Thai-
Burmese) borders, rendering them established multidrug-
resistant (MDR) areas. Chloroquine resistance spread
across Africa during the 1980s, and severe resistance
is especially found in east Africa. As a result, more than
ten African countries have switched
their first-line drug to sulfadoxine-
pyrimethamine. Of great concern is the
fact that the efficacy of this drug in Africa
is progressively deteriorating, especially
in foci in east Africa, which are classified
as emerging MDR areas. Urgent efforts
are needed to lengthen the lifespan
of sulfadoxine-pyrimethamine and to
identify effective, affordable, alternative
antimalarial regimens. Molecular markers
for antimalarial resistance have been
identified, including pfcrt polymorphisms
associated with chloroquine resistance and dhfr and dhps
polymorphisms associated with sulfadoxine-pyrimethamine
resistance. Polymorphisms in pfmdr1 may also be
associated with resistance to chloroquine, mefloquine,
quinine, and artemisinin. Use of such genetic information
for the early detection of resistance foci and future
monitoring of drug-resistant malaria is a potentially useful
epidemiological tool, in conjunction with the conventional
in-vivo and in-vitro drug-sensitivity assessments. This
review describes the various features of drug resistance in
Plasmodium falciparum, including its determinants, current
status in diverse geographical areas, molecular markers,
and their implications.
Lancet Infect Dis 2002; 2: 20918
Drug resistance in malaria is a vitally important public-
health concern. Each year, an estimated 0727 million
people die of malaria, and over 75% of them are African
children.
1
Amid such a disease burden, the development of
resistance has a significant influence on the control of
malaria in affected countries.
Resistance of Plasmodium falciparum to chloroquine, a
4-aminoquinoline, was first observed almost 50 years ago.
Today, chloroquine resistance occurs almost everywhere
that P falciparum does. Strains of falciparum parasites have
developed resistance to most of the commonly used
antimalarials, including sulfadoxine-pyrimethamine
(Fansidar) and mefloquine. Resistance commonly develops
within 1015 years after an antimalarial is introduced
(table 1).
26
The development of resistance to sulfadoxine-
pyrimethamine in P falciparum in Africa is particularly
serious, because this drug combination is the only
affordable, effective, practical, and well-tolerated alternative
to 4-aminoquinolines.
We review here the changing patterns of drug resistance
and our current understanding of its development,
including molecular clues that have recently been elucidated.
The resistance pattern in each geographical region provides
useful treatment guidance, because there are no bedside
methods for assessment of antimalarial-drug susceptibility.
Although drug resistance occurs in both P falciparum and
Plasmodium vivax, only the former is discussed in this paper
because it accounts for most of the disease burden. We
emphasise those areas where multidrug resistance is
emerging. Resistance to chloroquine in P vivax largely
concentrates in Papua New Guinea and Irian Jaya
(Indonesia) with sporadic reports from elsewhere in Asia
and South America. No resistance has been documented for
Plasmodium malariae or Plasmodium ovale, the other species
that infect human beings.
THE LANCET Infectious Diseases Vol 2 Xxxxxx 2002 http://infection.thelancet.com 209
Epidemiology of drug-resistant malaria
Chansuda Wongsrichanalai, Amy L Pickard, Walther H Wernsdorfer, and Steven R Meshnick
CW is an epidemiologist at the Armed Forces Research Institute of
Medical Sciences (AFRIMS), Bangkok, Thailand. ALP is a doctoral
student and SRM is professor of epidemiology at the Department of
Epidemiology, University of North Carolina School of Public Health,
Chapel Hill, NC, USA. WHW is professor at the Department of
Specific Prophylaxis and Tropical Medicine, Institute of
Pathophysiology, University of Vienna, Vienna, Austria.
Correspondence: Dr Chansuda Wongsrichanalai, AFRIMS, 315/6
Rajvithi Road, Bangkok 10400, Thailand. Tel +66 (0) 2 644 5775;
fax +66 (0) 2 644 4784; email chansudaw@thai.amedd.army.mil
Reviews
Table 1. Dates of introduction and first reports of antimalarial drug resistance
Antimalarial drug Introduced First reported Difference Refs
resistance (years)
Quinine 1632 1910 278 2
Chloroquine 1945 1957 12 3
Proguanil 1948 1949 1 2,4
Sulfadoxine-pyrimethamine 1967 1967 0 2,4
Mefloquine 1977 1982 5 5
Atovaquone 1996 1996 0 6
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THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 210
Historical perspectives
Quinine is one of the oldest malaria remedies known,
although its use has never been as widespread as that of
more contemporary drugs. Quinine occurs naturally in the
bark of cinchona trees in South America. Cinchona bark was
introduced into Europe as a treatment for the ague in the
early 17th century. In 1820, the alkaloid quinine was isolated
from cinchona bark.
7,8
Chloroquine was first synthesised in Germany but was
not recognised as a potent antimalarial until the 1940s as
part of the US World War II military effort. By 1946, it was
found to be far superior to other contemporary synthetic
antimalarials.
9
It became the cornerstone of antimalarial
chemotherapy for the next 40 years. However, the advent of
chloroquine resistance led to the development of other
drugs such as mefloquine, sulfadoxine-pyrimethamine,
artemisinin derivatives, and atovaquone-proguanil
(Malarone).
7,8
Subsequently, resistance has developed to
many of the newer drugs.
In China, infusions prepared from wormwood
(Artemisia annua) were used for treating fever over a
thousand years ago. The efficacy of the infusions has been
ascribed to the sesquiterpene lactone, artemisinin. As a
result of increasing drug-resistant malaria, artemisinin from
wormwood and semisynthetic derivatives of this substance
have become a very important antimalarial drug group.
Assessment of P falciparum antimalarial
susceptibility
P falciparum susceptibility to antimalarial drugs is
commonly assessed by therapeutic response (in-vivo test).
In-vivo response to drugs was originally defined by WHO in
terms of parasite clearance (sensitive [S] and three degrees of
resistance [RI, RII, RIII]).
10
This classification remains valid
for areas with low or no malaria transmission, but it is
difficult to apply to areas with intensive transmission, where
new infections may be mistaken for recrudescences.
Therefore, WHO introduced in 1996 a modified protocol
based on clinical outcome (adequate clinical response, early
treatment failure, and late treatment failure) targeted at a
practical assessment of therapeutic responses in areas with
intense transmission, where parasitaemia in the absence of
clinical signs or symptoms is common.
10,11
The protocol has
also been adapted for use in areas of low to moderate
endemicity, taking into consideration that the objectives of
malaria treatment are both parasite clearance and
disappearance of symptoms. A summary of the original and
modified protocols is shown in table 2. If not otherwise
indicated, the term resistance in this review is based on
therapeutic response according to one of these established
protocols.
Antimalarial-drug susceptibility can also be assessed
by in-vitro assays measuring the intrinsic sensitivity of
P falciparum from the inhibition of growth or schizont
maturation.
3,11
Recently, the use of molecular markers has
been proposed as an additional tool for the early detection of
drug resistance in malaria.
12
Each assessment method has its
advantages and disadvantages, and the results may not be
directly comparable with each other. In-vitro test results, in
particular, do not necessarily correspond to in-vivo
outcomes, largely owing to the role of host immunity in the
latter. In addition, pharmacokinetic information may be
required for differentiation between true resistance and
failure to achieve adequate drug concentration profiles.
13
Determinants of antimalarial resistance
Many factors contribute to the development and spread of
resistance. Gene mutations conferring resistance to
antimalarial drugs do occur in nature, independently of
drug effect (the common antimalarial drugs are not
mutagenic). Although the natural proportion of such
mutants in the parasite population is low, and malaria
isolates from populations and
individuals show heterogeneity,
selection of the most fit parasites
occurs under drug pressure. Single or
multiple point mutations in the
plasmodium genome may confer
resistance in the face of chemotherapy.
Reasons for the development and
spread of drug resistance involve
the interaction of drug-use patterns,
characteristics of the drug itself, human
host factors, parasite characteristics,
and vector and environmental factors
(table 3).
3,5,1416
Characteristics of the drug are
important determinants of resistance.
First, drugs with a long elimination
half-life, such as mefloquine, may exert
substantial residual selection on new
infections contracted after the
treatment of the primary infection
when the drug persists at
subtherapeutic concentrations in the
Review
Epidemiology of drug-resistant malaria
Table 2. Classifications of in-vivo antimalarial-drug sensitivity test outcomes
according to the original WHO protocol and the modification (1996) for areas with
substantial malaria transmission
10
Classification Definition
Original classification
S (sensitive) Reduction to <25% of initial parasitaemia on day 2 with smears
negative for malaria from day 7 to the end of follow-up (28 days or
longer for drugs with a long half-life, such as mefloquine)
RI response Initial clearance of parasitaemia, a negative smear on day 7, followed
by recrudescence 8 or more days after treatment
RII response Initial clearance or substantial reduction of parasitaemia (<25% of
the initial count on day 2) but with persistence or recrudescence of
parasitaemia during days 47
RIII response No significant reduction of parasitaemia
Modified classification (1996)
Early treatment failure (ETF) Aggravation or persistence of clinical symptoms in the presence of
parasitaemia during the first 3 days of follow-up
Late treatment failure (LTF) Reappearance of symptoms in the presence of parasitaemia during
days 414 of follow-up
Adequate clinical response (ACR) Absence of parasitaemia on day 14 irrespective of fever, or absence
of clinical symptoms irrespective of parasitaemia, in patients not
meeting ETF or LTF criteria
For personal use. Only reproduce with permission from The Lancet Publishing Group.
THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 211
plasma,
16
especially in areas with
intense malaria transmission. Second,
the maintenance of adequate drug
concentrations over a long enough
time is important for clearing the entire
population of parasites within a
given individual. Subtherapeutic drug
concentrations eliminate the most
susceptible parasites and leave those
that may be more fit to recover and
reproduce. As a result, the necessary
therapeutic dose may increase beyond
the maximum tolerated, and manifest
drug resistance will emerge. Third,
widespread use of drugs at high
intensity serves to increase drug
pressure and is a determinant
for selection of resistant parasite
populations.
More potent immune responses
increase the efficacy of chemotherapy.
A semi-immune patient might be cured
by a drug despite the fact that his
parasites are partially drug resistant.
Individuals who are naive to malaria
generate a non-specific immune
response that is not as effective as the
specific immunity elicited by repeated
infections. Thus, the introduction of
resistant malaria into non-immune populations such as refugees
or migrants increases the opportunity for manifestation and
spread of resistance, because parasites with low or moderate
resistance would be cleared in semi-immune populations.
Level of transmission influences the rate of development
and spread of drug resistance but its exact role is complex and
is most probably multifactorial. Increased risk of drug-
resistance development has been postulated to occur in areas
of both low
17
and high
15
transmission. The general
observations that resistance developed earlier in areas of low
transmission (such as Thailand and Brazil) and is still more
prevalent in such areas than in those with higher transmission
tend to support the low-transmission hypothesis. As an
example of the high transmission hypothesis, full chloroquine
resistance in children occurred and spread within 25 years in
an area of high transmission in east Africa that had been
under massive chloroquine pressure.
18
Finally, vector and environmental factors may influence the
proliferation of resistant parasites. For example, chloroquine-
resistant parasites may be more fit for reproduction in certain
anopheline mosquitoes than non-resistant strains.
16
Description of drug resistance worldwide
Chloroquine
Chloroquine resistance has been reported from wherever
falciparum malaria is endemic, except Central America and
the Caribbean.
3,10
In the late 1950s, resistance to chloroquine
was noted on the Thai-Cambodian border and in Colombia.
3
All endemic areas in South America were affected by 1980 and
almost all in Asia and Oceania by 1989.
In Africa, chloroquine resistance was first documented
in the east in 1978. Resistance spread to the central and
southern parts of the continent before arriving in west Africa
in 1983. By 1989, chloroquine resistance was widespread in
sub-Saharan Africa. Extensive reviews of the spread of
chloroquine resistance have been published by Wernsdorfer
and Payne
3
and Peters.
2
In general, resistance is currently less
severe in west and central Africa than in east Africa, but even
in west Africa, its intensity varies from an advanced stage
with severe effects on mortality and morbidity in focal areas
of Senegal,
19,20
to a moderate degree in Ghana
21
and
Cameroon,
22
and a low level in Mali.
23
Owing to high-
intensity chloroquine resistance, more than ten countries
in Africa have already switched their first-line treatment
to sulfadoxine-pyrimethamine or a combination of
chloroquine and sulfadoxine-pyrimethamine.
Chloroquine-resistant parasites in Africa were thought
by some to share the same origin as the Indochina strains,
but by others to have developed locally as a result of mass
drug administration plus intrinsic entomological,
epidemiological, and parasitological factors that promoted
local resistance selection.
3,16
Current molecular studies
suggest the Asian origin of African isolates, but at least four
different foci of chloroquine resistance have so far been
identified.
12
Polymorphisms in two genes of the P falciparumgenome
are the focus of studies on the molecular basis of
chloroquine resistance (panel 1). The pfcrt gene
24
is located
on chromosome 7, and codes for PfCRT, a vacuolar
membrane transporter protein. Many polymorphisms that
Review
Epidemiology of drug-resistant malaria
Table 3. Determinants of antimalarial-drug resistance
Factor and characteristics Example
Drug
Half-life Resistance to LAPDAP (short half-life) develops more slowly than
that to sulfadoxine-pyrimethamine (long half-life)
14
Dosing Use of subtherapeutic doses in self-treatment such as with antifolate
drugs in Thailand in the 1970s; poor drug compliance; mass drug
administration with subtherapeutic doses; use of chloroquinised salt
3
Non-target drug pressure Presumptive use of antimalarial drugs without laboratory diagnosis
or for indications other than malaria
Pharmacokinetics Use of drug formulations with reduced bioavailability
Cross-resistance Sulfadoxine-pyrimethamine and sulfamethoxazole-trimethoprim
Human
Host immunity Non-immune, migrant gem-miners and resistance to mefloquine on
the Thai-Cambodian border
5
Maintenance of resistant Non-detection of drug failure
parasite reservoir
Parasite
Genetic mutations See panel 1
Transmission level Whether low or high transmission has more influence on drug
resistance is debatable; prevalence of drug resistance is higher in
regions of low transmission, whereas a model suggests the benefits
of transmission control in delaying resistance development
15
Vector and environment
Vector affinity of parasites Increased infectivity and productivity of chloroquine-resistant
parasites in Anopheles dirus and the propagation of chloroquine
resistance in southeast Asia and western Oceania
16
LAPDAP=chlorproguanil plus dapsone.
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THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 212
are associated with chloroquine resistance have been
identified, but the substitution of threonine for lysine in
codon 76 was recently shown in vitro to associate absolutely
with resistance in isolates from Africa, South America, Asia,
and Papua New Guinea.
23,25
Findings from an in-vitro study
of isolates from various origins
26
and several clinical studies
in diverse geographical areas support the association
between the Thr76 mutation and chloroquine resistance in
Africa (Mali,
23
Cameroon,
27
Sudan,
28
Mozambique
29
), Asia
(Laos,
30
Thailand
31
), and South America (Brazil
32
). However,
one study in Uganda, where chloroquine resistance is more
prevalent than in west Africa, reported no association.
33
Most of the studies reporting an association note that,
although the Thr76 mutation may be essential for the
resistant phenotype, it is also present to a lesser degree in
chloroquine-sensitive strains, suggesting that other
polymorphisms in pfcrt are necessary or that several genes
are involved. Additionally, host immunity is known to have
a significant role in parasite clearance, therefore chloroquine
treatment success in semi-immune individuals even in the
presence of chloroquine-resistant parasites with the Thr76
mutation is not unexpected. Djimde and colleagues
23
found
that the lack of the Thr76 mutation was highly predictive of
chloroquine treatment success, whereas its presence
accounted for only a third of treatment failures.
Another gene, pfmdr1, which is located on chromosome
5 and codes for P-glycoprotein homologue 1 (Pgh1), has
generated interest in resistance to chloroquine and other
antimalarials. The aspartic acid to tyrosine point mutation
in codon 86 has been associated with chloroquine resistance
in some clinical and in-vitro studies (Mali,
23
The Gambia,
34
Sudan,
28
Uganda,
35
Thailand,
36
Brazil
37
), but not in others
(Uganda,
33
Laos,
30
Thailand,
38
Brazil
39
). Several other pfmdr1
polymorphisms, notably Phe184, Cys1034, Asp1042, and
Tyr1246, have been implicated to varying degrees in
chloroquine resistance (table 4). Although evidence for the
association of pfmdr1 with chloroquine resistance has not
been as convincing as for pfcrt, a recent parasite transfection
experiment showed that polymorphisms in the pfmdr1 gene
modulate susceptibility to chloroquine (as well as to
mefloquine and the structurally related compounds quinine
and halofantrine).
49
Linkage disequilibrium between pfcrt Thr76 and pfmdr1
Tyr86 has been noted in Africa (Nigeria
50
and Sudan
28
) and
provides support for a convergence of polymorphisms
necessary for a resistance phenotype. The slow development
of chloroquine resistance may signal such a multifactorial
mechanism.
12
Sulfadoxine-pyrimethamine
Resistance to sulfadoxine-pyrimethamine was first noted on
the Thai-Cambodian border in the mid-1960s,
4
and it
became an operational problem in the same area within a
few years of its introduction to the malaria-control
programme in 1975.
51
Currently, high-level resistance is
found in a large part of southeast Asia, southern China, and
the Amazon Basin.
10,46,52
Lower degrees and frequencies of
resistance are observed on the Pacific coast of South
America, southern Asia east of Iran, and western Oceania.
11
In Africa, sulfadoxine-pyrimethamine sensitivity started
declining in the late 1980s. Resistance is rapidly gaining
ground on this continent, more so in the east than in the
west. In east Africa, the degree of resistance is variable. High
percentages of RII/RIII responses were documented in
children in an endemic area of Tanzania as early as 1994.
53
This finding was thought to be attributable to previous drug
pressure due to the use of pyrimethamine-dapsone
prophylaxis. The 19992000 data from the East African
Network for Monitoring Antimalarial Treatment indicated
that the proportion of clinical failures (combined late and
early treatment failures) at some sentinel sites in Kenya was
already more than 25%, and the proportion of
parasitological failures at day 7 in children has reached 45%
at one site in Tanzania. Focal areas of low to moderate
sulfadoxine-pyrimethamine resistance exist throughout
Africa.
21,5456
Resistance is likely to progress geographically
and in its intensity at an alarming rate if nothing is done to
interrupt its course.
An in-vitro study in Kenya showed low sensitivity to
both pyrimethamine and sulfadoxine from the late 1980s to
the early 1990s and the presence of pyrimethamine-resistant
isolates in the late 1980s, even before sulfadoxine-
pyrimethamine was widely used.
57
In-vitro sulfadoxine-
pyrimethamine resistance was documented across sub-
Saharan Africa, varying in prevalence from 1330%: in
Tanzania in 1994,
58
Equatorial Guinea in 199092,
59
Gabon
in 1994,
60
and Ghana in 1991.
21
Cross-resistance between
trimethoprim and pyrimethamine in P falciparum was
recently demonstrated.
61
Therefore, widespread use of
trimethoprim-sulfamethoxazole for prophylaxis against
opportunistic in people with AIDS in Africa might further
shorten the useful lifespan of sulfadoxine-pyrimethamine.
The molecular basis of resistance to sulfadoxine-
pyrimethamine is the best characterised of all antimalarial
resistance. Specific mutations in P falciparum that lead to
resistance to both sulfadoxine and pyrimethamine have
Review
Epidemiology of drug-resistant malaria
Panel 1. Molecular markers of antimalarial
resistance
Chloroquine
pfcrt Thr76 strongly associated
pfmdr1 Tyr86, Phe184, Cys1034, Asp1042, Tyr1246 possibly
associated
Sulfadoxine-pyrimethamine
Principal mutations in codons 108, 51, 59, and 164 of dhfr
gene and codons 436, 437, 540, 581, and 623 of dhps gene
dhfr Asn108 essential for pyrimethamine resistance
Degree of resistance increases with additional mutations,
Leu51 and Arg59, or triple mutation
Absolute resistance conferred by the addition of Leu164, thus
quadruple mutation, irrespective of dhps mutations
Mefloquine, quinine
Genetic basis of resistance not yet clearly understood
pfmdr1 polymorphism may modulate mefloquine and quinine
susceptibility
Point mutations being explored at positions 86, 184, 1034,
1042, and 1246 of pfmdr1.
Artemisinin
No clinically relevant resistance as yet.
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THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 213
been identified. Sulfadoxine and pyrimethamine act
synergistically. The former inhibits dihydropteroate
synthetase (DHPS) and the latter inhibits dihydrofolate
reductase (DHFR). These two enzymes are involved in folate
synthesis.
Point mutations in the following five codons of the dhps
gene known to date are implicated in conferring resistance
by decreasing the binding affinity of the enzyme (table 4):
serine at codon 436 to alanine or phenylalanine; alanine at
437 to glycine; lysine at 540 to glutamic acid; alanine at 581
to glycine; alanine at 613 to serine or threonine. Gly437 and
Glu540 have been reported to occur
together or singly in various parts of
the world (Indonesia,
40
Vietnam,
42
Malawi, Kenya, Bolivia,
44
and
Gabon
43
). Gly581 has been observed in
South America alone or with
Gly437.
44,45
Specific point mutations in the
dhfr gene known to be associated with
pyrimethamine resistance by
reduction in drug-binding affinity of
DHFR include: alanine at 16 to valine,
asparagine at 51 to isoleucine, cysteine
at 59 to arginine, serine at 108 to
asparagine or threonine, and
isoleucine at 164 to leucine (table 4).
This combination of mutations has
been observed in Thailand, where
high-level sulfadoxine-pyrimethamine
resistance is well recognised. The triple
mutation of Ile51, Arg59, and Asn108
has been observed in Vietnam
42
and
east Africa (Kenya and Malawi).
44
Asn108 and Arg59 have been observed
in combination in Indonesia.
40
Asn108, Ile51, Leu164, accompanied
by mutation of cysteine at 50 to
arginine and a repeat between codons
30 and 31 were also noted in Bolivia
44
and Brazil,
46
areas where sulfadoxine-
pyrimethamine resistance is prominent
in South America. Arg50, in addition
to Asn108 and Ile51, was also
associated with sulfadoxine-
pyrimethamine resistance in
Venezuelan isolates by Urdaneta and
colleagues,
45
who noted that mutations
in both genes (Asn108 and Ile51 in
dhfr and Gly437 and Gly581 in dhps)
might be necessary for sulfadoxine-
pyrimethamine resistance in that
region. Leu164 has so far been
reported only from areas with high-
level sulfadoxine-pyrimethamine
resistance (southeast Asia,
42
Bolivia,
44
and Brazil
46
). The Ala16, Thr108
genotype is known to be specific for
cycloguanil resistance and does not
confer resistance to pyrimethamine.
62
Overall, a change from serine at codon 108 to asparagine
is known to be the key mutation for pyrimethamine
resistance. Additional point mutations in three other
codons, Ile51, Arg59, and Leu164, are known to increase
progressively the degree of resistance. Thus, quadruple
mutants (those with the Leu164 mutation) confer the most
severe resistance, more severe than triple mutants (those
with Ile51 and Arg59). Although the precise relation
between mutations in the dhfr and dhps genes in clinical
sulfadoxine-pyrimethamine resistance is unclear, current
Review
Epidemiology of drug-resistant malaria
Table 4. Drug-specific polymorphisms reported in P falciparum
Drug and polymorphism Location Refs
Chloroquine
pfcrt
Thr76, Ile74, Glu75 Asia, Africa 26
Thr76 Africa 28
Thr76 Africa 27
Thr76, Ile74, Glu75, Ser220, Ile371 Africa 23
Thr76, Ser72 (one isolate) S America 26
pfmdr1
Tyr86 Asia 36
Tyr86, Asp1042 Indonesia 40
Tyr86 Africa 28
Tyr86 Africa 34
Tyr86 Africa 41
Tyr86, Asp1042, Tyr1246 Africa 35
Tyr86, Asp1042, Tyr1246, Phe184, Cys1034 S America 37
Asp1042, Tyr1246 S America 39
Sulfadoxine-pyrimethamine
dhps
Gly437, Glu540 Asia 42
Gly437, Glu540 Indonesia 40
Gly437 Africa 43
Glu540 Africa 44
Gly437, Gly581 S America 45
Gly581 S America 44
Gly437, Glu540, Gly581 S America 46
dhfr
Asn108, Ile51, Arg59 Asia 42
Asn108, Arg59 Indonesia 40
Asn108, Ile51, Arg59 Africa 44
Asn108, Arg50, Ile51, Leu164, Bolivia repeat S America 44
Asn108, Arg50, Ile51, S America 45
Asn108, Arg50, Ile51, Leu164, Bolivia repeat S America 46
Mefloquine
pfmdr1
Tyr86* Thailand 36
Copy number Thailand 36
Copy number Thailand 47
Tyr86* Africa 48
Quinine (decreased sensitivity)
pfmdr1
Tyr86, Phe184, Cys1034, Asp1042, Tyr1246 S. America 37
Tyr86 Africa 48
Artesunate
pfmdr1
Copy number Thailand 36
Dihydroartemisinin
pfmdr1
Tyr86* Africa 48
* Increases sensitivity.
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THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 214
data show that the presence of a sensitive dhfr allele is highly
predictive of sulfadoxine-pyrimethamine treatment success
irrespective of the dhps allele.
62
Quinine
Although recrudescence strongly suggestive of quinine
resistance was noted in Brazil nearly 100 years ago,
successive observations of clinical resistance to quinine only
began to accumulate during the mid-1960s, especially from
the Thai-Cambodian border.
2
Currently, clinical resistance
to quinine monotherapy occurs sporadically in southeast
Asia and western Oceania. Data from in-vitro assays indicate
that resistance is less frequent in South America
37
and
Africa.
63
Widespread use of quinine in Thailand in the early
1980s as an interim therapy in the face of declining
sulfadoxine-pyrimethamine efficacy resulted in significant
reduction of its sensitivity.
16
Therefore, for the past two
decades, quinine has been consistently used in combination
with a partner antibiotic such as tetracycline or doxycycline
to increase the effectiveness of treatment. Quinine is
presently reserved as a second-line or third-line drug and is
used in cases of severe malaria.
There is some suggestion that pfmdr1 mutations
associated with chloroquine resistance may also account for
reduced susceptibility to quinine. In a Brazilian study of
pfmdr1 mutations (Asn86, Phe184, Cys1034, Asp1042,
Tyr1246), chloroquine-resistant strains were found to have
low susceptibility to quinine.
37
In The Gambia, pfmdr1 Tyr86
was weakly associated with decreased sensitivity.
48
Mefloquine
Mefloquine resistance was first observed near the Thai-
Cambodian border in the late 1980s.
5,64
The advent of
mefloquine resistance in Thailand may have been influenced
by the heavy use of the chemically related drug quinine just
before the introduction of mefloquine.
16
Mefloquine alone is
no longer effective on the Thai-Myanmar and Thai-
Cambodian borders, although it is still operationally useful
in most other endemic areas in and around Thailand with
field efficacy of more than 75%. By in-vitro assays, resistant
strains are common in the neighbouring countries.
65
Migrant gem-miners returning from Cambodia may have
been the means of the spread of mefloquine resistance to
Bangladesh and India.
16
There are also case-reports of mefloquine resistance
from the Amazon Basin, but the degree and scope of
resistance in South America are still far below those
of southeast Asia. In-vitro studies suggest the presence
of P falciparum strains with low mefloquine sensitivity
in Africa,
63,66
but clinical mefloquine resistance is rare
in Africa.
Copy number and polymorphisms of the pfmdr1 gene
have been investigated as molecular markers of mefloquine
resistance. The evidence on increased pfmdr1 copy number
as a molecular marker for mefloquine resistance remains
conflicting. Two studies in Thailand suggested that a
higher copy number confers mefloquine resistance,
36,47
but
other studies did not confirm that finding (Thailand,
38
Brazil,
37
Africa
41
).
Some studies have shown increased sensitivity to
mefloquine with the pfmdr1 Tyr86 mutation (The Gambia,
48
Thailand
36
), suggesting a possible inverse relation between
sensitivity to mefloquine and to chloroquine.
48
Reed and
colleagues
49
showed in a parasite transfection experiment
that Ser1034, Asn1042, and Asp1246 mutations were the
cause of resistance to mefloquine (as well as the structurally
related drugs quinine and halofantrine). These findings
strengthen the role of pfmdr1 as the key modulator of
mefloquine resistance.
Artemisinin
Artemisinin and derivatives (eg, artemether, artesunate, and
dihydroartemisinin) are associated with a high rate of
recrudescences after monotherapy, probably because of the
pharmacodynamic properties of these agents.
13
Therefore,
artemisinins are not used alone for clinical treatment; they
are usually combined with longer-acting antimalarials such
as mefloquine or lumefantrine. Adequate dose regimens of
combinations of artemisinins and mefloquine or artemether
and lumefantrine have not been shown to produce
treatment failures due to artemisinin resistance. On the
whole, both in-vivo and in-vitro sensitivity testing of
P falciparum in the multidrug-resistance area of the Thai-
Myanmar border, where the combination of artesunate and
mefloquine has been the first-line regimen for the past 7
years, remains satisfactory (Thai Ministry of Public Health
statistics). Except in an animal model, so far there has not
been any solid evidence of artemisinin resistance.
The development of artemisinin resistance may be
delayed by the characteristics of this drugnamely, short
elimination half-life and ability to reduce gametocyte-
carriage rate.
13
In mice, artemisinin resistance has been
associated with diminished drug uptake and the
overexpression of a potential target protein, the
translationally controlled tumour protein.
67
A molecular
study has suggested that the pfmdr1 Tyr86 variant may also
be associated with increased sensitivity to artemisinin.
48
The
transfection study mentioned above also showed that
Ser1034, Asn1042, and Asp1246 mutations additionally alter
the sensitivity of P falciparumto artemisinin.
49
Multidrug resistance
Multidrug resistance of P falciparum was defined previously
as resistance to more than two operational antimalarial
compounds of different chemical classes.
16
For the purpose
of this review, the definition is slightly modified by further
specifying the extent of resistance to a third drug class.
Generally, the first two classes are 4-aminoquinolines
(eg, chloroquine) and antifolates (eg, sulfadoxine-
pyrimethamine). Areas where the third drug is no longer
operationally effective are classified as having established
multidrug resistance. Although the precise criterion for the
loss of operational usefulness has not yet been clearly
defined, 25% treatment failure is widely quoted as the
maximum tolerated before the treatment policy should be
changed.
10
The figure shows the established multidrug-resistant
areas together with those where resistance to both
Review
Epidemiology of drug-resistant malaria
For personal use. Only reproduce with permission from The Lancet Publishing Group.
THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 215
chloroquine and sulfadoxine-pyrimethamine has been
documented, which thus have the potential for multidrug
resistance to emerge.
Established multidrug resistance
Established multidrug resistance occurs mainly in southeast
Asia, particularly the border regions of Thailand. Since 1995,
high-level mefloquine resistance led to replacement of this
drug alone with the combination of artesunate and high-
dose mefloquine for microscopically confirmed cases of
falciparum malaria in established multidrug-resistant areas
of Thailand. These areas include Maesod on the western
border with Myanmar and an area on the southeastern
border with Cambodia.
5
One study conducted in a confined
population near Maesod suggested that use of artemisinin
derivatives in combination with mefloquine might have
reduced mefloquine resistance.
68
Two important issues are
of particular relevance. First, the efficacy of this therapeutic
regimen must be maintained, because no practical
alternative to artemisinin is yet available. Second, multidrug
resistance must be limited to its present area. Self-treatment
with artesunate and mefloquine is common outside
Thailand, thus posing a threat of further resistance spread in
the region as a result of drug pressure.
Some focal areas in the Amazon Basin may also fall into
this category (figure). These areas have sporadic reports of
mefloquine resistance and reduced susceptibility to quinine.
Artesunate-mefloquine combination therapy is being
adopted as the first-line regimen on the border of Peru and
Brazil.
Emerging multidrug resistance
Areas are classified as having emerging multidrug
resistance on the basis of the widespread loss of clinical
efficacy of chloroquine and sulfadoxine-pyrimethamine,
representing drugs of the first two chemical classes, and the
potential for emergence of resistance to a third class of
drug. Because the third drug has not been widely used, its
efficacy is still maintained, especially in semi-immune
individuals. These areas are epidemiologically most
important, because they are generally associated with more
intense malaria transmission and less effective control
measures.
By definition, these seem to be limited to areas of east
Africa (such as Tanzania and Kenya) and the Amazon Basin
with high-degree sulfadoxine-pyrimethamine resistance.
However, in practice, precise boundaries of emerging
multidrug resistant areas are difficult to determine because
of the lack of reliable drug-susceptibility surveillance data.
Therefore, all areas with signs of progressive loss of
sulfadoxine-pyrimethamine efficacy are considered at risk of
emergence of multidrug resistance.
Progression to severe or complicated manifestations
and increased mortality associated with sulfadoxine-pyri-
methamine resistance have not yet been reliably assessed in
tropical Africa, but they are likely to be substantial in the
absence of affordable alternative medication. Measures to
counteract this threat are urgently needed. The selection of
the next operational drug after the loss of efficacy of the
two cheapest, chloroquine and sulfadoxinepyrimethamine,
is difficult.
Review
Epidemiology of drug-resistant malaria
Malaria transmission areas
Chloroquine resistance
Sulfadoxine/pyrimethamine reistance
Multidrug resistance
Malaria-free islands
Areas with reduced susceptibility of P falciparum to chloroquine and sulfadoxine-pyrimethamine (SP) and areas designated as multidrug resistant
according to WHO. Reproduced from map 00344 (WHO/CDS/RBM/2001.33)
10
with updated data and slight modification by colouring.
For personal use. Only reproduce with permission from The Lancet Publishing Group.
THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 216
Drug combinations offer a logical, albeit temporary,
solution.
13,69
The artesunate-mefloquine combination
currently used in the established multidrug-resistant areas of
Thailand cannot be expected to offer similarly favourable
operational prospects for Africa, where malaria epidemiology
and peripheral health-care systems in remote areas differ
substantially from those in Thailand.
70,71
In fact, at the cost of
US $35 per artesunate-mefloquine treatment (330 times
more than medication with sulfadoxine-pyrimethamine
or chloroquine), economic constraints prohibit this regimen
for Africa.
Future prospects
There are two issues particularly relevant to the future
scenario of drug-resistant malarianew antimalarial
regimens and use of molecular information for the control of
parasite drug resistance (panel 2).
New antimalarial regimens
Besides strengthening of overall malaria-control strategies,
current efforts have focused on the use of combination
chemotherapy in dealing with multidrug-resistant malaria.
Experience with drugs for treatment of HIV infection and
tuberculosis points to the importance of combination
chemotherapy in delaying the onset of resistance, because
the parasite has to mutate in several sites simultaneously
to become resistant to a combination, an event that is
much less likely than a single mutation conferring resistance
to single drugs. In malaria, options are combinations
of existing agents (chloroquine plus sulfadoxine-
pyrimethamine, artesunate plus sulfadoxine-pyri-
methamine, artesunate plus mefloquine, chlorproguanil
plus dapsone [LAPDAP]), and those involving new agents
(artemether plus lumefantrine [Coartem, Riamet]).
However, the efficacy of combinations involving old drugs
may be short-lived, since resistance-conferring mutations
already exist.
LAPDAP may be an affordable alternative to
sulfadoxine-pyrimethamine in semi-immune
individuals in Africa.
14
Like sulfadoxine-
pyrimethamine, both chlorproguanil and dapsone
are antifolates, but they have shorter half-lives than
sulfadoxine or pyrimethamine. Therefore the use
of LAPDAP is likely to delay the development of
resistance. Besides, LAPDAP is also effective
against infections associated with triple mutants in
dhfr that normally do not respond to sulfadoxine-
pyrimethamine therapy. Unfortunately, strains of
P falciparum with high-degree resistance to
sulfadoxine-pyrimethamine, such as those with
quadruple mutants in dhfr, are also resistant to
LAPDAP. Several other combination regimens are
undergoing field testing, especially in Africa, to
find out which would be the most effective and
appropriate for a given specific endemic region.
10
Malarone is a combination of atovaquone, a
new antimalarial drug of the naphthoquinone
class, and proguanil. Atovaquone disrupts electron
transport in the mitochondria of parasites, but it
does not affect the mitochondria of the human host. It was
first shown to be effective against P falciparum in vitro and
was subsequently found to be effective in treating patients
harbouring resistant organisms.
6
A recent study in Tanzania
showed its efficacy in the treatment of sulfadoxine-
pyrimethamine-resistant cases.
72
When the drug was used
alone, however, resistance developed rapidly. It is therefore
partnered with proguanil, with which atovaquone shows
synergy,
73
in the hope that atovaquone resistance may be
delayed. The molecular mechanism of resistancemutations
in cytochrome bwas first identified in Pneumocystis carinii
and was later found to apply for malarial parasites as well.
74
The high cost of Malarone, however, will make it unrealistic
for use in Africa.
14
Use of molecular information
In the near future, more alleles that lead to drug resistance
are likely to be identified, and the full genome sequence of
P falciparum will be elucidated. This valuable genetic
information should be used efficiently, for example, to
identify new drug targets, construct an effective vaccine, or
design a tool for rapid diagnosis of antimalarial resistance.
Some genetic markers are potentially useful for the
surveillance of drug resistance.
12
They can also be used in
support of in-vitro and in-vivo studies such as the
application of pfcrt Thr76 mutation in predicting
chloroquine treatment outcomes.
23
Conclusions
Drug resistance is probably the greatest challenge that
most malaria-control programmes are facing. Although
the problem of drug-resistant malaria is worldwide,
Africa is of greatest concern. Development of resistance
to chloroquine in P falciparum forced several African
countries to rely on sulfadoxine-pyrimethamine as the
first-line drug. Loss of sulfadoxine-pyrimethamine
sensitivity is progressing rapidly, especially in parts
Review
Epidemiology of drug-resistant malaria
Panel 2. Future concerns and prospects
Areas with no drug-resistant malariaCentral America north of
Panama Canal and the island of Hispaniola (Haiti and Dominican Republic)
G Efforts needed to extend chloroquine efficacy as long as possible.
Areas with no multidrug resistanceMost of Africa, parts of South
America, and parts of Asia
G Constant surveillance needed for the early detection of development
of multidrug resistance
Emerging multidrug-resistant areasFoci in tropical Africa, many
areas of South America, southern Asia east of Iran, and western Oceania
G Prevention and rapid detection of new foci development
G New regimens and alternative drugs needed
G Shifting to drugs other than 4-aminoquinolines and antifolates
is expensive
G Rational drug use must be reinforced to limit drug pressure
Established multidrug-resistant areasSoutheast Asia with
concentration in the western and eastern borders of Thailand, foci in the
Amazon Basin
G Efforts needed to limit geographical spread
G New combinations may preserve efficacy of current drugs
G The development of new drug candidates needed
For personal use. Only reproduce with permission from The Lancet Publishing Group.
THE LANCET Infectious Diseases Vol 2 April 2002 http://infection.thelancet.com 217
of east Africa, rendering them potentially emerging
multidrug-resistant areas. Given the limited resources
for other malaria-control measures, rational drug use
is critical, although this is known to be hampered by
economic constraints and scarcity of drug choices.
There is no over-riding answer to the challenge of
multidrug-resistant malaria. The useful lifespan of a new
antimalarial may be lengthened if it is used in combination
with other drugs, and therefore combination therapy
has been the focus for the management of multidrug-
resistant malaria. The advantages and drawbacks of
each candidate regimen must be carefully considered
for specific endemic areas. Todays drug choice may affect
future drug policies and the ability to prevent epidemics and
to control morbidity and mortality due to malaria.
Review
Epidemiology of drug-resistant malaria
Search strategy and selection criteria
We identified the data for this review by searches of Medline,
WHO documents, and books that have been cited in previous
publications by authorities on malaria chemotherapy. Search
terms were: malaria, drug resistant, chemotherapy, and
epidemiology. Only papers published in English were
included.
The past decade has brought progress in the
understanding of the molecular basis of drug resistance
in P falciparum. Sets of mutations in dhfr and dhps
genes have been implicated as the causes of sulfadoxine-
pyrimethamine resistance. The pfcrt and pfmdr1 genes
are the focus of studies on resistance to other drugs. The
former is strongly associated with chloroquine resistance,
and polymorphisms in the latter may alter the parasites
susceptibility to mefloquine, halofantrine, quinine, and
artemisinin as well as chloroquine. Overall, the right
combination of point mutations in specific falciparum
genes may be necessary for a drug-resistant phenotype.
As more information on the genetics of antimalarial
resistance becomes available, design of new, improved,
molecular-based tools for early detection and interventions
that aim at limiting the extent of established multidrug
resistance and preventing new foci of drug resistance from
emerging may become possible.
Acknowledgments
The authors work was supported by the US Department of
Defense Global Emerging Infections Surveillance (GEIS) Program
and NIH grant AI 45426. We thank G Dennis Shanks for comments.
The views of the authors do not purport to reflect those of the US Army
or US Department of Defense.
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Review
Epidemiology of drug-resistant malaria
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