Next

Volume 352:1565-1577 April 14, 2005 Number 15

Effectiveness of Antimalarial Drugs

J. Kevin Baird, Ph.D.

The opening of the Panama Canal in 1914 initiated an era in which efforts to control malaria were aimed at the anopheline mosquito.
The World Health Organization's Global Malaria Eradication Campaign in the 1950s and 1960s marked the apex of these efforts. The
use of dichlorodiphenyltrichloroethane (DDT) rid vast areas of endemic malaria virtually everywhere except in sub-Saharan Africa.
The eradication strategy was abandoned in 1969, because it came to be considered logistically, socially, and politically impractical,
especially given public concern about the effects of DDT on the environment. It took two decades and a global resurgence of malaria
before a new strategy emerged, one that was focused on treatment rather than prevention.1 This treatment strategy is currently being
debated, and its efficacy is unproved.2,3 Both the broad collapse of preventive efforts and the waning efficacy of standard antimalarial
drugs4,5 account for the global resurgence of malaria. New therapies are available, but the use of older drugs persists for social,
economic, and clinical reasons, despite resistance of the organisms to the older drugs. Efforts to use new antimalarial drugs may be
hampered by regulatory requirements or economic obstacles as well as by important questions about safety for the large numbers of
patients who treat themselves. Public health agencies continue to support the distribution of older therapies, despite strong criticism
from scientists conducting research on the disease and its treatment and prevention.6 In addition to inadequate drug efficacy, new
therapies may fail because of inappropriate use, inadequate absorption, poor adherence, contraindications, intolerability, the use of
counterfeit drugs or improper manufacture of drugs, or prohibitive cost. Chloroquine and sulfadoxine–pyrimethamine, which for
several decades were the foundation of malaria therapy, had a low risk with respect to this array of problems. However, the
emergence and consolidation of resistance to these drugs eroded their clinical usefulness.

Plasmodia

The coccidian genus plasmodium contains 172 species that infect birds, reptiles, and mammals. Four species infect humans —
Plasmodium falciparum, P. malariae, P. ovale, and P. vivax. P. falciparum and P. vivax account for the vast majority of the 300 million
to 500 million infections that occur each year. Endemic transmission takes place in most tropical latitudes and reaches into temperate
zones seasonally (Figure 1). Although each type of infection causes debilitating febrile illness, only P. falciparum carries a substantial
risk of death; 1 million to 3 million deaths occur each year in regions of holoendemic infection in sub-Saharan Africa, most of them
among infants and young children. For plasmodia, humans are not the "definitive host," a concept defined by parasitologists as the
site where sexual recombination occurs. Instead, many species in the genus anopheles have that distinction. From the perspective of
the parasite, humans are simply a means of getting into mosquitoes, where sexual recombination can occur. A full description of the
extraordinary complexity of this cycle is beyond the scope of this review. Figure 2 illustrates the essential features of the cycle and the
terminology of antimalarial therapies.

Figure 2. Antimalarial Drug Activity in the Life Cycle of Plasmodia.

Tissue-stage schizonticides kill the asexual stages developing in the liver, including liver schizonts (all species) and quiescent
hypnozoites (Plasmodium vivax and P. ovale), thus preventing primary or secondary attacks (relapses) of clinical malaria. Blood-
stage schizonticides interrupt asexual schizogony (mitotic division) in red cells, preventing or terminating clinical attacks of malaria.
Gametocytocides kill or sterilize sexual stages in the blood, thus preventing infection of mosquitoes and transmission of the disease.
Another class of drugs, the sporontocides (which kill forms developing in the mosquito, including the sporozoites that infect humans),
is not represented here, because none are available for clinical use.

Extrinsic Determinants of Drug Effectiveness

Availability

Most developing nations license and make available only the antimalarial drugs that are provided through national health programs.7
This approach often excludes relatively expensive or risky therapies, even for patients who may be able to afford a given drug and
have access to medical supervision. The main factor affecting availability is economic — the ability or inability to purchase a drug for
broad distribution and the potential reluctance to use an agent because of an inability either to screen users or to monitor its quality.
Chloroquine and sulfadoxine–pyrimethamine cost less than 15 percent of the cost of the least expensive alternative agents and
approximately 1 to 2 percent of the cost of many of the agents marketed in the developed world (Table 1). The developing world
requires distribution strategies for effective therapies that overcome the availability of cheap but ineffective drugs. The U.S. National
Academies made a core recommendation8 that governments and international financial institutions should commit $300 million to
$500 million per year within five years to subsidize artemisinin-combined therapies (an emerging family of antimalarial drugs, including
artemether, artesunate and dihydroartemisinin) to achieve prices for the end user in the range of 10 to 20 cents. Unless this
recommendation is followed, market forces will continue to drive the use of ineffective therapies.

Adherence

Most people taking antimalarial drugs live in rural regions of the developing world and are not supervised by health professionals. A
study conducted among 1640 febrile patients with malaria in Burkina Faso9 showed that 69 percent were self-treated, and in a study
in Ethiopia, among 630 febrile patients with malaria, 67 percent were self-treated.10 Complex, inconvenient, or poorly tolerated
antimalarial regimens carry a substantial risk of inadequate adherence.11 Among 414 Brazilian patients, the risk of recurrent malaria
correlated with self-reported poor adherence,12 whereas among 632 Nigerian children strict adherence correlated with clinical
recovery.13 Convenient and easily understood packaging and education of the patients alleviate poor adherence. Owing to lengthy
and complex regimens, currently used therapies such as quinine and primaquine and new combined therapeutic strategies14
challenge the ease of adherence.

Counterfeit and Substandard Drugs

Counterfeit antimalarial drugs pose a serious threat in regions where the trade in pharmaceuticals is not rigorously regulated. A
survey conducted in Cameroon found insufficient or inactive ingredients in 38 percent of preparations labeled chloroquine, 78 percent
of those labeled quinine, and 12 percent of tablets labeled as an antifolate agent.15 A survey in Southeast Asia involving 104
purchases of artesunate tablets found that 38 percent of the tablets contained no drug.16 The trade in counterfeit drugs undoubtedly
results in many deaths, but it is lucrative and carries little risk of imprisonment.17 The inadvertent marketing of substandard
pharmaceuticals poses another threat. In a survey of eight authorized wholesalers in Tanzania selling combined sulfadoxine–
pyrimethamine tablets, 11 percent of the tablets failed industry standards for content, and 44 percent failed dissolution testing.18

Intrinsic Determinants of Drug Effectiveness

Stage Specificity

Plasmodia pass through distinct stages of form, function, location, clinical consequence, and susceptibility to antimalarial drugs
(Figure 2 and Table 1). Drug activity ranges from narrow (e.g., the activity of quinine against asexual blood stages) to broad (e.g., the
activity of primaquine against sexual and asexual forms in the blood and liver). Moreover, the range of stage-specific susceptibility
differs among species of plasmodia — for example, chloroquine kills the gametocytes of P. vivax but exerts no effect against those of
P. falciparum. These intrinsic properties define the recommended uses of antimalarial drugs.

Parasite Burden

Most patients with malaria carry a burden of 108 to 1013 parasites.19 Effective chemotherapy induces a constant fractional decline with
each asexual cycle,20 at a rate that varies according to the susceptibility of the parasite to a given drug. For example, artemisinin
derivatives induce reductions of 104, whereas tetracycline achieves a reduction by only a factor of 10 with each cycle. The duration of
exposure to a drug that is needed to eliminate infection hinges on the intrinsic rate of decline and, more important, on the initial
parasite burden.21 High levels of parasitemia, as compared with a low burden, require longer exposure to effective drug levels and
have a relatively higher risk of treatment failure.22

Pharmacokinetics

A variety of factors affect the pharmacokinetics of antimalarial drugs. Variant alleles of the human gene for cytochrome P-450 2C19
(CYP2C19), for example, correlate with slow or rapid metabolism of some antimalarial agents. For example, chloroguanide,
metabolized through the activity of CYP2C19, was studied in 126 healthy, unrelated Nigerian subjects, of whom 5 percent had slow
metabolism of the drug (8 percent of the normal rate).23 A study of Thai subjects taking oral contraceptives reported that metabolism of
chloroguanide was decreased by 34 percent,24 a finding that may be explained by inhibition of CYP2C19 activity by estrogen. Certain
foods may also profoundly affect the bioavailability (and toxicity) of certain antimalarial drugs — for example, fatty foods affect the
bioavailability of halofantrine and of the combination of lumefantrine and artemether.

Safety and Tolerability

In rural health care centers, staff with relatively little formal training may be responsible for taking care of patients with malaria. The
patients often have no written medical history, and the centers have minimal capacity for laboratory screening for contraindications to
particular medications. Worse still, most of these people with malaria treat themselves with antimalarial agents acquired over the
counter or passed from household to household.

Any antimalarial drugs that are to be distributed in such regions need to be safe and well tolerated, and the risks associated both with
the indicated use and with contraindicated or inappropriate uses should be considered. Clinical studies supporting the licensing of
new antimalarial drugs rarely include highly vulnerable groups such as infants, young children, and pregnant women.25 Thus, safety
issues often drive the decision to continue distributing chloroquine or sulfadoxine–pyrimethamine, despite substantial parasite
resistance. Key data with regard to the safety and tolerability of the most commonly used antimalarial drugs are summarized in Table
2.

Immunity

The intersection of immunity to plasmodia and antimalarial drug activity is poorly understood. In a study of Brazilian patients, self-
reported poor adherence correlated with a risk of recurrence among nonimmune persons (i.e., those with a first infection) but not
among semi-immune persons (i.e., those with >4.8 years' exposure to plasmodia).12 Among 80 Lao patients, the level of humoral
immunity to P. falciparum and the risk of therapeutic failure were correlated with the parasite count on admission to the hospital and
the length of time to the resolution of fever.29 Djimde et al.30 showed that the clearance of P. falciparum organisms carrying a mutant
transporter gene, pfcrt K76T, that was linked with resistance to chloroquine correlated with acquired immunity in subjects in sub-
Saharan Africa. In subjects with relatively little immunity who were infected with mutant parasites, the chloroquine therapy failed more
frequently than in those infected with the same genotype of P. falciparum organisms who had clinical immunity. However, such
correlations do not directly link immune effectors with drug activity; acquired immunity independently reduces the parasite burden,
which is a well-known determinant of antimalarial effectiveness.

The acquired immunity that occurs throughout regions of holoendemic infection in sub-Saharan Africa may explain the relatively late
increase in resistance in that region. Most people at risk in South America and Asia lack immunity and consequently carry
substantially higher parasite burdens than do most Africans. Moreover, their symptomatic infections are generally treated far more
often than are asymptomatic infections among Africans. In Asia and South America, these factors substantially increase the
probability of the selection of resistant genotypes.14
Resistance

The parasites causing malaria exhibit a range of susceptibility to antimalarial agents. Several distinct phenomena explain this range,
including species-specific innate resistance (e.g., asexual blood stages of P. falciparum lack susceptibility to primaquine, whereas
those of P. vivax appear to be sensitive to it31); strain-specific innate resistance (e.g., that of asexual liver stages of P. vivax from the
island of New Guinea against primaquine32); and acquired resistance. Of these, acquired resistance is the most important, because
failure may occur even in the presence of complete adherence to the recommended therapies. Studies in Senegal suggest that
resistance to chloroquine contributed to an increase by a factor of 2 to 11 in mortality from malaria.33 In western Kenya, case fatality
rates were markedly higher among children receiving chloroquine than among those receiving other therapies.34

Most of the data on the responses of P. falciparum to therapy reflect this emphasis on acquired resistance. Nonetheless, P. vivax
profoundly affects public health outside Africa,35 and data are also available that cover its resistance to chloroquine. P. malariae and
P. ovale infect relatively few people, and little is known about acquired resistance in these species. Resistance to chloroquine in P.
malariae was reported from Indonesia36 but is apparently debatable.37

Genetic mutations among strains of plasmodia have been linked with phenotypes of clinical resistance. Some studies challenge
specific links between genotype and phenotype in certain settings, but putative markers have emerged (Table 3); the extensive
literature on this topic has been reviewed. 38,39,40,41,42,43 Validated genetic determinants of resistance would vastly improve surveillance
and, thus, the effectiveness of resources for the treatment and control of malaria.

Drug Resistance

The risk of resistance varies according to species, strain, and drug. Estimates of risk are often confounded by the determinants
already described. Methods of estimating risk also vary. The following estimates of the risk of treatment failure were distilled from
many studies and should not be construed as quantitative region-specific risks, because patterns of resistance vary tremendously,
even within nations. Protocols that evaluate efficacy generally follow the guidelines of the World Health Organization, which
recommend a follow-up period of 7, 14, or 28 days.44 The timing of recurrent parasitemia or disease reflects the degree of resistance
— earlier failure represents higher-grade resistance. Late recurrence may be confounded by reinfection, especially in regions of
intense transmission. Investigators in sub-Saharan Africa thus favor an in vivo test with a duration of 7 or 14 days, whereas those in
other regions tend to favor a 28-day test or even 63-day test. Comparison of genotypes of the strains causing original and recurrent
parasitemias can be used to address confounding by reinfection, but relatively few reporting clinics or laboratories have the capacity
to conduct such testing. Tests conducted in Africa tend to report both parasitologic failure (i.e., recurrent parasitemia after treatment,
independent of clinical presentation) and the clinical failure of treatment, whereas investigators elsewhere focus on parasitologic
failure.44

Chloroquine

After an analogue that had been developed in Germany was captured in 1943, during World War II, chloroquine quickly came into
universal use as therapy for and prophylaxis against malaria.45 Chloroquine was highly effective, easily administered, and inexpensive
and had good safety and tolerability. However, resistance in P. falciparum appeared in the late 1950s in Thailand and Colombia and
emerged in the 1970s in New Guinea and eastern sub-Saharan Africa. Today, resistance to chloroquine in malaria caused by P.
falciparum occurs everywhere except in Central America (and Hispaniola) and in some regions of southwestern Asia.

In sub-Saharan Africa, the risk of parasitologic treatment failure with the use of chloroquine was almost uniformly greater than 40
percent, whereas the risk of clinical treatment failure tended to be higher in the eastern and central portions of the African continent
(typically >30 percent and often >50 percent, respectively) than in the west (typically <20 percent).8 Elsewhere, the rates of
parasitologic failure at day 28 were 57 percent (of 209 evaluations) in southwestern Asia,46,47,48,49,50 46 percent (2280 evaluations) in
southern Asia,51,52,53,54,55,56,57 85 percent (223 evaluations) in Southeast Asia,58,59,60,61,62,63,64,65,66 and 66 percent (137 evaluations) in South
America.67,68,69,70,71 Prescribing chloroquine monotherapy against this parasite in any setting, except one in which its effectiveness has
recently been demonstrated, should be considered irresponsible.

Resistance to chloroquine by P. vivax has emerged, apparently having originated in New Guinea, where failure rates now approach
100 percent.63 In contrast, surveys in Thailand reveal uniformly sensitive vivax malaria (not shown in Figure 1).72 Chloroquine-resistant
P. vivax may be characterized as endemic to the Indonesian archipelago, especially in the east (including New Guinea), sporadic in
the rest of Asia, and rare in South America.73

Sulfadoxine–Pyrimethamine

Sulfadoxine–pyrimethamine, which has potent efficacy against chloroquine-resistant and pyrimethamine-resistant P. falciparum,
became available in 1971 and became the standard second-line therapy against chloroquine-resistant falciparum malaria. This
combination acts synergistically against folate synthesis, inhibiting dihydropteroate synthase and dihydrofolate reductase. Although
rare, idiosyncratic allergic reactions have occurred among users of sulfa drugs, sulfadoxine–pyrimethamine has otherwise offered
superior safety and tolerability, along with the advantage of single-dose therapy. However, resistance to sulfadoxine–pyrimethamine
was recognized at the Thai–Cambodian border in the 1960s, and failures occurred in refugee camps in Thailand in the 1970s.38

The rates of parasitologic failure of treatment with sulfadoxine–pyrimethamine in sub-Saharan Africa were relatively high in southern
regions of holoendemic infection (>50 percent) and low elsewhere (<5 percent, except in Cameroon, where the rate of failure was 10
percent). The rates of clinical treatment failure in sub-Saharan Africa were similarly distributed (<5 percent in the west and 8 to 34
percent in the east and south).8 Studies in Southeast Asia indicated that the rates of parasitologic failure at day 7 and day 28 were 36
percent and 49 percent, respectively.74,75,76,77,78,79 Good efficacy (80 percent) persisted elsewhere — in southwestern Asia and on the
Horn of Africa, where no parasitologic failures were reported among 362 evaluations80,81; in southern Asia, where the failure rate was
18 percent (of 339 evaluations) by day 2855,56,57; and in South America, where the failure rate was 9 percent (of 50 evaluations) by day
7, 14 percent (of 42 evaluations) by day 14, and 6 percent (of 119 evaluations) by day 28.82,83,84 The risk of resistance to sulfadoxine–
pyrimethamine is relatively high in Southeast Asia and eastern Africa.

Mefloquine
Mefloquine emerged as a successor to chloroquine in the 1980s. Resistance appeared in regions at the border between Thailand and
Cambodia within a few years, perhaps owing to widespread use of quinine, to which it is structurally related.38 Resistance in that
border region remains high.85 However, in nearby regions resistance to mefloquine remains relatively low. Smithuis et al.62 reported
that, of 75 patients, more than 90 percent in the region of the western border of Myanmar (Burma) responded to mefloquine (at a dose
of 15 mg per kilogram of body weight). In another study, of 79 patients hospitalized in Bangkok and treated with mefloquine (25 mg
per kilogram), 68 (86 percent) remained free of parasitemia after 28 days.86 Similar efficacy was observed in Bangladesh.56 However,
a group of Dutch marines in Cambodia who were receiving mefloquine prophylaxis during the 1990s had high attack rates of
falciparum malaria.87 In contrast, the protective efficacy of mefloquine among Indonesian soldiers in western New Guinea — where
the efficacy of chloroquine against P. falciparum and P. vivax approaches zero63 — was 100 percent.88 The risk of the prophylactic or
therapeutic failure of mefloquine in Southeast Asia appears to be low outside the shared borders of Thailand, Myanmar, and
Cambodia.

Few studies have evaluated the effectiveness of mefloquine against falciparum malaria in Africa. In the mid-1990s, Lobel et al.89
examined mefloquine prophylaxis among 140 Peace Corps volunteers who were infected by P. falciparum. Poor adherence explained
most of the infections, but in five cases the resistance appeared to be genuine (i.e., parasitemia was present with >620 ng of
mefloquine per milliter in plasma). Despite sporadic, well-documented failures of mefloquine among travelers to Africa, the drug
remains effective there.

Studies conducted in coastal Peru and in the Amazon Basin among 153 subjects with P. falciparum malaria who were receiving
mefloquine (at a dose of 15 mg per kilogram) revealed complete sensitivity.68,90,91 Despite occasional reports of resistance in the
Amazon basin, the available evidence shows a low risk of resistance throughout South America.

Mefloquine has received adverse attention in the media in recent years. The drug has been blamed for suicides, homicides, and other
personal tragedies on the basis of anecdotal accounts, which, by their nature, cannot establish cause and effect. Well-controlled trials
consistently indicate that mefloquine given as prophylaxis is as well tolerated as other antimalarial drugs.92 Nonetheless, the drug has
been linked with a higher risk of insomnia, fatigue, and adverse neuropsychiatric effects (e.g., depression and anger) than other
antimalarial drugs.93,94,95 The risk appears highest among women, especially those taking the drug for the first time and those with a
low body-mass index.96

Quinine

Jesuit priests in Peru in the 1500s learned from the Incas that powder from the bark of the cinchona tree relieved shivering with cold,
and they supposed it would also offer relief from the chills of malaria. Its activity against the parasite, later shown to be due to quinine,
was unknown to them. Resistance to quinine appears sporadically, and a moderate risk of treatment failure appears to be limited to
some regions of Southeast Asia and New Guinea. Zalis et al.97 suggested that there might be a link between poor in vitro responses
to quinine and diminished clinical responsiveness in the Amazon basin, but without supporting clinical data. Recent well-controlled
trials of quinine monotherapy showed variable rates of efficacy against P. falciparum: 92 percent in a group of 49 patients in
Bangladesh,56 67 percent among 54 patients in western Thailand, 98 and 80 percent among 30 patients in a clinic in Bangkok.99 A
seven-day regimen was more than 95 percent efficacious in Venezuela100 and Equatorial Guinea.101 Rare reports of failure of
intravenous quinine for the treatment of severe or complicated malaria have appeared sporadically.102

Oral quinine is used to treat uncomplicated malaria, generally over a period of three to seven days in combination with another blood-
stage schizonticide, typically tetracycline or doxycycline. Although one study of 86 patients in Thailand showed the superiority of a
seven-day regimen combined with tetracycline (a 100 percent efficacy rate) as compared with a five-day regimen combined with
tetracycline (87 percent),103 studies performed elsewhere have shown complete efficacy with shorter regimens combining quinine,
doxycycline, and primaquine.88,104

Poor adherence carries a high risk of treatment failure, particularly because quinine causes a syndrome of adverse effects known as
cinchonism, including primarily tinnitus, nausea, and vertigo. A randomized trial in Thailand recorded a 71 percent rate of adherence.11
However, in Cambodian villages, the rate of compliance with the same regimen was far lower — 11 to 20 percent — even after an
intervention to raise awareness about the need for compliance.105

Primaquine

Developed during World War II, primaquine remains the only licensed tissue-stage schizonticide (Figure 2) for the prevention of
relapse after infection (standard therapy) or as presumptive therapy against relapse after exposure to the risk of infection, without
evidence of infection (terminal prophylaxis). Recently recommended for use as prophylaxis,106 primaquine has a uniquely broad
spectrum, killing liver stages, asexual blood stages (of P. vivax31 but not P. falciparum107), and sexual blood stages. In endemic
regions, primaquine is widely used as a gametocytocide to prevent infection of mosquitoes.

Despite more than 50 years of use in millions of people per year, primaquine is still shrouded in confusion and genuine mystery. At
least four different regimens, some prescribed for only one week and others for as long as eight weeks, are aimed at the same
objective of preventing relapse.108 Three factors largely explain the lack of uniformity: first, the reluctance of health care programs to
accept therapy that has a duration of two weeks; second, perceived issues of toxicity and tolerability; and third, the use of a total dose,
rather than a dosing schedule, as the primary determinant of efficacy. How the same total dose of a rapidly eliminated drug kills
organisms irrespective of whether it is delivered over a period of 7, 14, or 56 days defies explanation.

Resistance is not fully understood either. Resistance in asexual blood stages of P. vivax has long been known109 but is of no clinical
consequence. Reports of resistance to tissue-stage schizonticidal activity fail to consider or describe patients' adherence, to exclude
the possibility of reinfection, or to address the possible recrudescence of chloroquine-resistant strains. Lack of evidence of resistance
to primaquine in liver stages probably reflects a heavy burden of proof rather than an absence of resistance.108

Combined Therapies against Resistance

Combined therapies, which constitute a widely practiced strategy in the treatment of diseases such as leprosy, tuberculosis, and
infection with the human immunodeficiency virus and which have long been known to be effective against malaria,110 have rarely been
used in its treatment until recently.111 The use of more than one agent successfully requires separate mechanisms of action against
the same stage of the parasite. Thus, because both sulfadoxine and pyrimethamine are folate antagonists, they would not be
considered a combined therapy. Similarly, combining a blood-stage schizonticide with primaquine is not considered combined
therapy, because the drugs attack different stages of the parasite. The fixed combination of atovaquone–chloroguanide, which affects
mitochondrial electron transport and folate metabolism in asexual blood stages, represents true combination therapy.112

Combined antimalarial therapies include old and new drugs — old drugs in new combinations (chloroquine and sulfadoxine–
pyrimethamine), an old drug combined with a new drug (amodiaquine and artesunate), and new drugs in combination (lumefantrine
and artemether).113,114 Combination therapy that includes artemisinin appears to be potent and particularly useful in endemic regions;
extracted from the weed Artemesia annua, the artemisinins (primarily artemether, artesunate, and dihydroartemisinin) were developed
in China during the 1960s.

The artemisinins act very rapidly, reducing parasitemia by a factor of 104 with each cycle. Thus, for a parasite burden in the range of
1012, only three cycles are required to abolish parasitemia. The artemisinins are rapidly eliminated, and daily administration for a
period of seven days (three cycles) is required. Episodes of recrudescence follow briefer regimens, but a seven-day regimen is
considered to be impractical. Therefore, treatment for a period of three days with artemisinin combined with a slowly eliminated
companion blood-stage schizonticide has been adopted. This three-day regimen of artemisinin reduces the parasite burden by a
factor of 108 (leaving only 0.000001 percent of parasites surviving to be abolished by mefloquine) (Figure 3). The artemisinins also
exert activity against gametocytes, reducing the probability of transmission.

Figure 3. Combination Therapies with Artemisinin Derivatives.

The figure illustrates the rationale behind the use of an artemisinin derivative combined with a slowly excreted drug such as
mefloquine for therapy against falciparum malaria over a period of five weeks. Panel A shows plasma levels of mefloquine (blue line)
and relatively high levels of parasitemia caused by strains resistant to mefloquine (solid red line) and sensitive to mefloquine (dashed
red line). Prescribed therapy eliminates the sensitive but not the resistant infection. Panel B shows the importance of parasite burden
as a determinant of drug effectiveness, indicating the elimination of both sensitive and resistant infections at lower levels of burden.
Panel C shows the quick inhibitory effect of a short regimen of an artemisinin derivative (black line), but, owing to its short plasma
half-life, the drug cannot completely eradicate the parasite. Panel D shows the effect of combining an artemisinin derivative (black
line) with a drug such as mefloquine (blue line): a quick "knockdown" of the parasite burden by the artemisinin derivative allows the
slower-acting and more slowly excreted mefloquine to exert activity against a greatly diminished parasite burden.

The use of combination therapies with artemisinin does not preclude the onset of drug resistance, particularly since patients may not
take the medication as directed. Thus, the multiple doses needed with such combined therapies (typically six doses over a period of
three days) represent an important potential pitfall. The emergence of resistant strains may already be in progress. In vitro studies
indicate diminished susceptibility to artesunate among Asian isolates.41 Isolates from western Cambodia, where combination therapies
with artemisinin derivatives are widely used, show diminished susceptibility to the combination of mefloquine and artesunate, as
compared with isolates from eastern Cambodia.115 In contrast, in the 1990s in some regions bordering western Thailand, combination
therapies with artemisinin derivatives were deployed as first-line therapy with excellent efficacy (more than 90 percent), and this
success appears to be stable.116 Where more resources are available, including medications, resistant strains have been slower to
develop. Where both medication and an effective health care infrastructure are present, resistance can be controlled. Such economic
and personnel issues may explain the contrasting findings with regard to rates of resistance to combination therapies with artemisinin
derivatives.117 Thus, supplying these therapies in the setting of an adequate health care infrastructure would seem the best way to
prevent the onset of resistance.

Patients with severe or complicated malaria often cannot take oral medication, and parenteral quinine has been the standard
approach to therapy. Rectal artesunate may radically improve the care of such patients, especially across the geographic expanses of
regions of endemic infection where parenteral medications are neither available nor practical. Clinical trials indicated that rectal
artesunate (combined with another blood-stage schizonticide) cleared parasitemia more quickly than parenteral quinine and with
equal efficacy.118,119

Summary

A global public health threat due to the resurgence of malaria stems from a general collapse of vector-control operations and from
resistance to chloroquine or sulfadoxine–pyrimethamine. Recent surveys show rates of treatment failure higher than 50 percent for
chloroquine in most affected regions, as well as poor efficacy of sulfadoxine–pyrimethamine in sub-Saharan Africa and Southeast
Asia. Quinine and mefloquine remain effective therapies everywhere except in some regions bordering Thailand. Resistance to
primaquine — the only drug for preventing relapse — probably occurs but has not yet been confirmed. New drugs should be effective
among the poor, self-treating rural populations in regions of endemic disease and should be provided through programs that address
issues of availability and cost, convenience and adherence, safety and tolerability, and quality assurance. The combination therapies
with artemisinin derivatives represent the present best efforts toward providing such therapeutic agents. These drugs deliver an
inhibitory effect that substantially reduces the probability of selection for resistant parasites, as compared with traditional
monotherapies. However, widespread distribution without complementary capabilities in the delivery of health care places the clinical
usefulness of these critical drugs in doubt.

. References

1. Trigg PI, Kondrachine AV. Commentary: malaria control in the 1990s. Bull World Health Organ 1998;76:11-16. [ISI][Medline]

2. Roberts DR, Laughlin LL, Hsheih P, Legters LJ. DDT, global strategies, and a malaria control crisis in South America.
Emerg Infect Dis 1997;3:295-302. [ISI][Medline]

3. Curtis CF. Should the use of DDT be revived for malaria vector control? Biomedica 2002;22:455-461. [Medline]

4. Baird JK. Resurgent malaria at the millennium: control strategies in crisis. Drugs 2000;59:719-743. [ISI][Medline]
5. Guerin PJ, Olliaro P, Nosten F, et al. Malaria: current status of control, diagnosis, treatment, and a proposed agenda for
research and development. Lancet Infect Dis 2002;2:564-573. [CrossRef][ISI][Medline]

6. Attaran A, Barnes KI, Curtis C, et al. WHO, the Global Fund, and medical malpractice in malaria treatment. Lancet
2004;363:237-240. [CrossRef][ISI][Medline]

7. Goodman C, Kachur SP, Abdulla S, et al. Retail supply of malaria-related drugs in rural Tanzania: risks and opportunities.
Trop Med Int Health 2004;9:655-663. [CrossRef][ISI][Medline]

8. Arrow KJ, Panosian C, Gelband H, eds. Saving lives, buying time: economics of malaria drugs in an age of resistance.
Washington, D.C.: National Academies Press, 2004:12.

9. Muller O, Traore C, Becher H, Kouyate B. Malaria morbidity, treatment-seeking behavior, and mortality in a cohort of young
children in rural Burkina Faso. Trop Med Int Hlth 2003;8:290-6.

10. Deressa W, Ali A, Enqusellassi F. Self-treatment in rural communities, Butajira, southern Ethiopia. Bull World Health Organ
2003;81:261-268. [ISI][Medline]

11. Fungladda W, Honrado ER, Thimasarn K, et al. Compliance with artesunate and quinine + tetracycline treatment of
uncomplicated falciparum malaria in Thailand. Bull World Health Organ 1998;76:Suppl 1:59-66. [Medline]

12. Duarte EC, Gyorkos TW. Self-reported compliance with last malaria treatment and occurrence of malaria during follow-up in
a Brazilian Amazon population. Trop Med Int Health 2003;8:518-524. [CrossRef][ISI][Medline]

13. Okonkwo PO, Akpala CO, Okafor HU, Mbah AU, Nwaiwu O. Compliance to correct dose of chloroquine in uncomplicated
malaria correlates with improvement in the condition of rural Nigerian children. Trans R Soc Trop Med Hyg 2001;95:320-
324. [CrossRef][ISI][Medline]

14. White NJ. Antimalarial drug resistance. J Clin Invest 2004;113:1084-1092. [Abstract/Full Text]

15. Basco LK. Molecular epidemiology of malaria in Cameroon XIX. Quality of antimalarial drugs used for self-medication. Am J
Trop Med Hyg 2004;70:245-250. [Abstract/Full Text]

16. Newton P, Proux S, Green M, et al. Fake artesunate in southeast Asia. Lancet 2001;357:1948-1950. [CrossRef][ISI]
[Medline]

17. Newton PN, White NJ, Rozendaal JA, Green MD. Murder by fake drugs. BMJ 2002;324:800-801. [Full Text]

18. Minzi OM, Moshi MJ, Hipolite D, et al. Evaluation of the quality of amodiaquine and sulfadoxine/pyrimethamine tablets sold
by private wholesale pharmacies in Dar Es Salaam, Tanzania. J Clin Pharm Ther 2003;28:117-122. [CrossRef][ISI][Medline]

19. White NJ, Krishna S. Treatment of malaria: some considerations and limitations of the current methods of assessment.
Trans R Soc Trop Med Hyg 1989;83:767-777. [CrossRef][ISI][Medline]

20. Day NPJ, Pham TD, Phan TL, et al. Clearance kinetics of parasites and pigment-containing leukocytes in severe malaria.
Blood 1996;88:4694-4700. [Abstract/Full Text]

21. White NJ. Why is it that antimalarial drug treatments do not always work? Ann Trop Med Parasitol 1998;92:449-
458. [CrossRef][ISI][Medline]

22. Ittarat W, Pickard AL, Rattanasinganchan P, et al. Recrudescence in artesunate-treated patients with falciparum malaria is
dependent on parasite burden not on parasite factors. Am J Trop Med Hyg 2003;68:147-152. [Abstract/Full Text]

23. Bolaji OO, Sadare IO, Babalola CP, Ogunbona FA. Polymorphic oxidative metabolism of proguanil in a Nigerian population.
Eur J Clin Pharmacol 2002;58:543-545. [CrossRef][ISI][Medline]

24. McGready R, Stepniewska K, Seaton E, et al. Pregnancy and use of oral contraceptives reduces the biotransformation of
proguanil to cycloguanil. Eur J Clin Pharmacol 2003;59:553-557. [CrossRef][ISI][Medline]

25. Taylor WR, White NJ. Antimalarial drug toxicity: a review. Drug Saf 2004;27:25-61. [Medline]

26. Health information for international travel, 2003–2004. Atlanta: Centers for Disease Control and Prevention, 2003:99-116.

27. Phillips-Howard PA, Wood D. The safety of antimalarial drugs in pregnancy. Drug Saf 1996;14:131-145. [ISI][Medline]

28. Wernsdorfer WH. Coartemether (artemether and luemfantrine): an oral antimalarial drug. Expert Rev Anti Infect Ther
2004;2:181-96.
 29. Mayxay M, Newton PN, Khanthavong M, et al. Chloroquine versus sulfadoxine-pyrimethamine for treatment of Plasmodium
falciparum malaria in Savannakhet Province, Lao People's Democratic Republic: an assessment of national antimalarial
drug recommendations. Clin Infect Dis 2003;37:1021-1028. [CrossRef][ISI][Medline]

Am. J. Trop. Med. Hyg., 68(4), 2003, pp. 398-402

Copyright © 2003 by The American Society of Tropical Medicine and Hygiene

NEW HAPLOTYPES OF THE PLASMODIUM FALCIPARUM CHLOROQUINE

RESISTANCE TRANSPORTER (PFCRT) GENE AMONG CHLOROQUINE-
RESISTANT PARASITE ISOLATES
HADYA S. NAGESHA, GERARD J. CASEY, KARL H. RIECKMANN, DAVID J. FRYAUFF, BUDI S. LAKSANA, JOHN C.
REEDER, JASON D. MAGUIRE, AND J. KEVIN BAIRD
The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia; Eijkman Institute for Molecular Biology, Jakarta,
Indonesia; United States Naval Medical Research Unit No. 2, U.S. Embassy, Jakarta, Indonesia; Papua New Guinea Institute of
Medical Research, Goroka, Papua New Guinea; Australian Army Malaria Institute, Brisbane, Australia

ABSTRACT

Mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene were examined to assess their associations
with chloroquine resistance in clinical samples from Armopa (Papua) and Papua New Guinea. In Papua, two of the five pfcrt
haplotypes found were new: SVIET from Armopa and CVIKT from an isolate in Timika. There was also a strong association (P <
0.0001) between the pfcrt 76T allele and chloroquine resistance in 50 samples. In Papua New Guinea, mutations in the pfcrt gene
were observed in 15 isolates with chloroquine minimum inhibitory concentrations (MICs) of 16–64 pmol, while the remaining six
isolates, which had a wild-type pfcrt gene at codon 76, had MICs of 2–8 pmol. These observations confirm that mutations at codon 76
in the pfcrt gene are present in both in vivo and in vitro cases of chloroquine resistance, and that detection of the pfcrt 76T allele could
predict potential chloroquine treatment failures.

INTRODUCTION

Chloroquine has been the drug of choice for treating malaria patients for the last 50 years, but the spread of drug-resistant
Plasmodium falciparum has become a major problem. In Southeast Asia, 21.9 million cases of malaria were reported in 1995 alone.1,2
Malaria is a serious problem in the eastern islands of Indonesia and in nearby Papua New Guinea. Approximately 20–30% of the
population in these regions typically carry malaria parasites at any given time. In addition, 20% of consultations, 16% of hospital
admissions, and 14% of hospital deaths are attributable to malaria.1,2
Papuan Indonesia (formerly Irian Jaya) and Papua New Guinea have long been plagued by drug-resistant malaria. Resistance to
pyrimethamine and chloroquine in the Arso-Waris-Upper Tor River areas of Papuan Indonesia is believed to have arisen in 1959–
1961 with the mass distribution of medicated chloroquine and pyrimethamine salts.3 Resistance of P. falciparum to chloroquine was
reported from Kalimantan in 1973 and from Papua in 1975.4,5 An increased risk of chloroquine resistance was reported from the
Jayapura region of Papua, Indonesia in the1980s.6,7 Surveys conducted during the 1990s showed high malaria prevalence rates (60–
92%) and levels of chloroquine treatment failure of up to 80% among indigenous and immigrant communities of northern and central
Papuan Indonesia.8–12 However, due to its safety profile, low cost, and relative success in treating mildly symptomatic malaria
infections among immune and semi-immune patients, chloroquine remains the treatment of choice for malaria, and no effective
alternative strategy has been developed. Molecular markers of drug resistance in P. falciparum could prove useful in defining the
intensity of resistance in an individual patient and the extent and severity of the problem in communities.
The aim of this study was to examine P. falciparum chloroquine resistance transporter (pfcrt) gene haplotypes in parasite isolates with
known in vivo or in vitro chloroquine resistance responses.

MATERIALS AND METHODS

The Armopa region is located on the northwestern coast of Indonesian Papua. Prior to Javanese transmigration in 1995, there were
small traditional villages and a single health clinic in Armopa. No mass treatment or prophylaxis was practiced before transmigrant
arrivals, and the indigenous people of Armopa did not use antimalarial drugs for prophylaxis. However, chloroquine, Fansidar®
(pyrimethamine and sulfadoxine) (F. Hoffmann La Roche, Basel, Switzerland), and quinine were presumably available for treatment of
clinical malaria. Transmigrants, primarily from malaria-free Java and Bali, had no previous exposure to malaria and no history of
antimalarial drug use before settling in Armopa. As per national health standards, they were given chloroquine for self prophylaxis
during their first three months after arrival; thereafter, treatment with only chloroquine was provided for uncomplicated cases of clinical
malaria. Chloroquine is widely used for treatment of clinical malaria among transmigrants and is dispensed through a health clinic in
each settlement.
Blood samples were collected in 1996–1999 from study volunteers who were immigrants to the Armopa SP1 and SP2 sites and had
no history of malaria or antimalarial drug use. Patients who were positive for malaria were treated at the health center with
chloroquine, Fansidar®, and quinine as the respective first-, second-, and third-line drugs for uncomplicated malaria as per the
Indonesian National Health Policy. Chloroquine was given at a dose of 10 mg/kg on the first day, followed by 5 mg/kg 12, 24, and 48
hours later. A single dose of Fansidar® (1 mg/kg of pyrimethamine and 20 mg/kg of sulfadoxine) was given if there was chloroquine
treatment failure. This study was carried out after obtaining informed consent from all adult participants and from parents or legal
guardians of minors, and was reviewed and approved by the Ethics Committees for Protection of Human Subjects at the Ministry of
Health, Republic of Indonesia, the U.S. Navy Medical Research Unit No. 2 (Jakarta, Indonesia), and The Walter and Eliza Hall
Institute of Medical Research (Melbourne, Australia) and the Papua New Guinea Medical Research Advisory Committee.

RESULTS

Of 85 patients, 21 (24.7%) cases cleared their parasitemias within 72 hours, had no recurrence during 28 days of follow-up, and were
classified as sensitive to chloroquine. Fifty patients had persistent or recurrent parasitemias and were classified as resistant to
chloroquine. Data from 15 patient samples were excluded from analysis due to incomplete clinical histories, intercurrent infections
with P. vivax, or an inability to amplify gene products. Samples were analyzed for mutations in the pfcrt gene after amplification by a
polymerase chain reaction (PCR) of DNA extracted from blood samples, followed by restriction fragment length polymorphism (RFLP)
analysis and DNA sequencing.13,14 The DNA from a drug-sensitive strain (D10) was used as a positive control to monitor PCR
conditions. As expected, the PCR product was amplified with wild-type alleles of the pfcrt gene. No PCR products were amplified in
negative controls.
The results of pfcrt mutational analysis of samples from 50 cases of chloroquine treatment failure are shown in Table 1 . All 50
chloroquine-resistant samples carried the mutant pfcrt 76T allele. No mutation was detected at codon 73, but variations were found at
codons 72, 74, and 75 in 50 samples: SVMNT (24), CVIET (11), CVMNT (9), and SVIET (6). Statistical analysis (chi-square test with
Yates’ correction) showed that the pfcrt mutation at codon 76 was strongly associated with chloroquine resistance (P < 0.0001).15
Among the 21 chloroquine-sensitive samples, only one carried a mutated pfcrt 76 allele.
TABLE 1
In vitro chloroquine responses and Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene haplotypes among malaria
patients in Armopa, Indonesian Papua*

Analysis of RFLP results from amplification of chloroquine-resistant P. falciparum laboratory strains K1, W2mef, VNS, 7G8, a new
isolate, 2300, from Timika on the southern coast of Indonesian Papua, and two isolates, F2382 and F1568, from Flores, Indonesia
showed mutations in the pfcrt gene (Table 2 ). Seven of these chloroquine-resistant laboratory strains showed three different pfcrt
haplotypes with mutations at codons 74, 75, and 76: CVMNT (7G8), CVIKT (2300), and CVIET (K1, W2 mef, VNS, F2382, and
F1568). The wild-type haplotype CVMNK was found in the chloroquine-sensitive control strain D10.
TABLE 2
In vitro chloroquine responses and Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene haplotypes among
laboratory strains and field samples*

The Wosera region of East Sepik province in Papua New Guinea is highly endemic for malaria. Mutation analysis of the genes
involved in chloroquine resistance from Papua New Guinea has shown the presence of P. falciparum isolates carrying the pfcrt
SVMNT haplotype, which is usually found in South American parasites, but not the CVIET haplotype of Southeast Asian isolates.16
The results of mutational analysis of 21 samples of P. falciparum obtained from malaria patients in Papua New Guinea are shown in
Table 2 . Fifteen of these samples with chloroquine minimum inhibitory concentrations (MICs) between 16 and 64 pmol had the 76T
allele. However, six isolates with MICs of 2–8 pmol had the wild-type pfcrt allele at codons 72, 73, 74, 75, and 76. There were three
pfcrt haplotypes among the 21 Papua New Guinea samples (Table 2 ). The wild-type haplotype CVMNK was observed in isolates
with MIC values of 2–8 pmol, while the chloroquine-resistant pfcrt haplotypes CVMNT and SVMNT were observed in samples with
MICs between 16 and 64 pmol.
DISCUSSION

Although chloroquine resistance in P. falciparum has been reported in Indonesia and Papua New Guinea since the early 1970s,
molecular analysis and in vitro/in vivo responses to antimalaria drugs have only recently been determined in this region. This study
analyzed the association of mutations in the pfcrt gene in samples of P. falciparum that had been characterized as chloroquine
sensitive or resistant by in vitro or in vivo tests.14 A strong association between mutations in the pfcrt gene and chloroquine resistance
(P < 0.0001) was observed in those samples from individuals who had chloroquine treatment failure in vivo or had displayed
chloroquine MICs of 16–64 pmol in the in vitro test.
Studies to elucidate the molecular and biochemical mechanism of resistance to chloroquine have been in progress for more than a
decade. Chloroquine resistance in P. falciparum involves decreased accumulation of the drug. However, the precise mechanism is
not known.17 Mutations in the P. falciparum multidrug resistance 1 (pfmdr1) gene were implicated and involvement of at least two
genes was hypothesized; mutations in both the pfcrt and the pfmdr1 genes appear to be necessary for resistance to chloroquine.13,18,19
A mutation in the pfcrt gene (located on chromosome 7) at codon 76, with a change from lysine to threonine, has been invariably
found in chloroquine-resistant strains and also in chloroquine-resistant field samples from Laos, Cameroon, Mozambique, Uganda,
and South America.13,20–27 Transformation of chloroquine-sensitive isolates with the Dd2 pfcrt gene sequence containing the 76T
mutation consistently produced chloroquine-resistant clones, and insertion of the wild-type pfcrt gene caused resistant isolates to
exhibit sensitivity to chloroquine.13 Studies on field isolates have shown the occurrence of three haplotypes of pfcrt gene alleles:
CVMNK among chloroquine-sensitive isolates, CVIET among chloroquine-resistant isolates from Southeast Asia and Africa, and
SVMNT among chloroquine-resistant isolates from South America and Papua New Guinea.13,16,28 The presence of the 76T pfcrt gene
mutation has been correlated with risk of therapeutic failure when malaria due to P. falciparum is treated with chloroquine.22,29,30
Recently, an analysis of the genetic mutations associated with chloroquine resistance in an area highly endemic for malaria (the
Wosera region of East Sepik province in Papua New Guinea) was also reported.16 All (100%) samples from treatment failures
(Indonesian Papua) and 67% of the isolates (Papua New Guinea) collected prior to treatment in the in vitro studies carry the mutated
pfcrt allele 76 (Tables 1 and 2 ).
In this study, analyses of known laboratory isolates that are resistant to chloroquine showed the presence of a mutation in the pfcrt
gene. Samples collected in Papua New Guinea for in vitro chloroquine susceptibility testing provide further support for our data from
clinical studies in Armopa. Although a mutated pfcrt codon 76 is invariably present in chloroquine-resistant isolates, comparison of
pfcrt haplotypes revealed some interesting features. In both Armopa (Papua) and Papua New Guinea, the CVMNK haplotype was the
wild type. In Papua New Guinea, the chloroquine-resistant haplotypes detected were SVMNT and CVMNT, as demonstrated in other
studies (Table 3 ).16,28 Interestingly, the pfcrt haplotypes CVIET (African, Southeast Asian) and SVMNT (South American) were also
detected in Papuan samples. In addition, two new pfcrt haplotypes were detected in Papua that have not been previously reported:
SVIET, which was found in clinical samples isolated from cases of treatment failure in Armopa and CVIKT, a haplotype found in
chloroquine-resistant laboratory strain 2300, which was isolated in 1985 in Timika, Papua. The presence of African, South American,
Southeast Asian, and two new chloroquine-resistant haplotypes in these regions raises the question of the evolution of these five
haplotypes. They may have evolved by sequential mutations of the gene in this region, where the parasite is widely circulated, or the
parasites with these haplotypes were transferred into this region. This speculation on the origin of haplotypes awaits detailed studies
with data from other loci in the genomes of P. falciparum isolates.

TABLE 3
Geographic distribution of Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene haplotypes among chloroquine-
resistant strains of P. falciparum*

In conclusion, our results support the hypothesis that the molecular basis of chloroquine resistance involves mutations in the pfcrt
gene and that detection of a mutated pfcrt allele 76 could predict potential chloroquine treatment failures.
Received September 9, 2002. Accepted for publication December 4, 2002.
Acknowledgments: We thank Alan Cowman for supervision and encouragement during the course of this study; Sangkot Marzuki,
Syafruddin, and Graham Brown for their encouragement and support; and Jenny Thompson and Deborah Baldi for providing the D10
parasite. We also thank the Ministry of Health, Republic of Indonesia for assistance in the collection of specimens in Armopa, Papua.
Financial support: This work was supported by the Australian and Indonesian Governments through the Australian Agency for
International Development and Bappenas, respectively, and the Global Emerging Infections Surveillance program of the U.S.
Department of Defense. Hadya S. Nagesha was supported by a Traveling Fellowship from the Wellcome Research Trust (United
Kingdom).
Disclaimer: The views of the authors expressed herein are their own and do not purport or reflect those of the U.S. Navy or the U.S.
Department of Defense.
Authors’ addresses: Hadya S. Nagesha and Gerard J. Casey, The Walter and Eliza Hall Institute of Medical Research, 1G Royal
Parade, Melbourne, 3050, Australia, Telephone: 62-3-934-52479, Fax: 62-3-934-70852, E-mail: hadya@wehi.edu.au and Eijkman
Institute for Molecular Biology, Jl Diponegoro 69, Jakarta 10430, Indonesia. Karl H. Rieckmann, Australian Army Malaria Institute,
Brisbane, Australia. David J. Fryauff, Budi S. Laksana, Jason D. Maguire, and J. Kevin Baird, United States Naval Medical Research
Unit No.2, U.S. Embassy, Jakarta, Indonesia. John C. Reeder, Papua New Guinea Instititute of Medical Research, Goroka, Papua,
New Guinea.
REFERENCES

1. WHO, 1987. World malaria situation 1985. World Health Stat Q 40: 142–170.[Medline]

2. WHO, 1997. Malaria in the South-East Asia Region. New Delhi: Regional Office for South-East Asia, World Health
Organization.

3. Meuwissen TJHE, 1964. The use of medicated salt in an antimalaria campaign in West New Guinea. Trop Geogr Med 16:
245–255.

4. Clyde DF, McCarthy VC, Miller RM, Hornick RB, 1976. Chloroquine-resistant falciparum malaria from Irian Jaya, Indonesian
New Guinea. Am J Trop Med Hyg 79: 38–41.

5. Ebisawa I, Fukuyama T, 1975. Chloroquine resistance of Plasmodium falciparum in West Irian and East Kalimantan. Ann
Trop Med Parasitol 69: 275–282.[ISI][Medline]

6. Hoffman SL, Campbell J, Rustama D, Dimpudus AJ, Surumpaet B, 1987. Pyrimethamine-sulfadoxine still effective against
Plasmodium falciparum in Jayapura, Irian Jaya: RI-type resistance in 2 of 18 patients. Trans R Soc Trop Med Hyg 81: 276–
277.[ISI][Medline]

7. Hoffman SL, Harun S, Campbell JR, Purnomo, Marwoto HA, Dimpudus AJ, Rustama D, Oetoma HS, Rai NK, Laughlin LW,
1984. Prolonged incubation improves the micro-scale in-vitro test for drug sensitivity of Plasmodium falciparum. Lancet 1: 7–
9.[ISI][Medline]

8. Baird JK, Basri H, Purnomo, Bangs MJ, Subianto B, Patchen LC, Hoffman SL, 1991. Resistance to chloroquine by
Plasmodium vivax in Irian Jaya, Indonesia. Am J Trop Med Hyg 44: 547–552.[ISI][Medline]

9. Baird JK, Wiady I, Fryauff DJ, Sutanihardja MA, Leksana B, Widjaya H, Kysdarmanto, Subianto B, 1997. In vivo resistance
to chloroquine by Plasmodium vivax and Plasmodium falciparum at Nabire, Irian Jaya, Indonesia. Am J Trop Med Hyg 56:
627–631.[ISI][Medline]

10. Fryauff DJ, Sumawinata I, Purnomo, Richie TL, Tjitra E, Bangs MJ, Kadir A, Ingkokusumo G, 1999. In vivo responses to
antimalarials by Plasmodium falciparum and Plasmodium vivax from isolated Gag Island off northwest Irian Jaya, Indonesia.
Am J Trop Med Hyg 60: 542–546.[Abstract/Free Full Text]

11. Gomez-Saladin E, Fryauff DJ, Taylor WR, Laksana BS, Susanti AI, Purnomo, Subianto B, Richie TL, 1999. Plasmodium
falciparum mdr1 mutations and in vivo chloroquine resistance in Indonesia. Am J Trop Med Hyg 61: 240–244.[Abstract/Free
Full Text]

12. Pribadi W, Sutanto I, Atmosoedjono S, Rasidi R, Surya LK, Susanto L, 1998. Malaria situation in several villages around
Timika, south central Irian Jaya, Indonesia. Southeast Asian J Trop Med Public Health 29: 228–235.[Medline]

13. Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW, Su
XZ, Wootton JC, Roepe PD, Wellems TE, 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein
PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6: 861–871.[ISI][Medline]

14. Nagesha HS, Din-Syafruddin, Casey GJ, Susanti AI, Fryauff DJ, Reeder JC, Cowman AF, 2001. Mutations in the pfmdr1,
dhfr and dhps genes of Plasmodium falciparum are associated with in vivo drug resistance in Irian Jaya, Indonesia. Trans R
Soc Trop Med and Hyg 95: 43–49.[ISI][Medline]

15. Dawson-Saunders B, Trapp RG, 1994. Basic and Clinical Biostatistics. Norwalk, CT: Appleton and Lange.

16. Mehlotra RK, Fujioka H, Roepe PD, Janneh O, Ursos LM, Jacobs-Lorena V, McNamara DT, Bockarie MJ, Kazura JW, Kyle
DE, Fidock DA, Zimmerman PA, 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in
association with pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci USA 98: 12689–12694.
[Abstract/Free Full Text]
17. Krogstad DJ, Gluzman IY, Kyle DE, Oduola AM, Martin SK, 1987. Efflux of chloroquine from Plasmodium falciparum:
mechanism of chloroquine resistance. Science 238: 1283–1285.[ISI][Medline]

18. Foote SJ, Kyle DE, Martin RK, Oduola AMJ, Forsyth K, Kemp DJ, Cowman AF, 1990. Several alleles of the multidrug-
resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 345: 255–258.[ISI][Medline]

19. Reed MB, Saliba KS, Caruana SR, Kirk K, Cowman AF, 2000. Pgh1 modulates sensitivity and resistance to multiple
antimalarials in Plasmodium falciparum. Nature 403: 906–909.[ISI][Medline]

20. Basco LK, Ringwald P, 2001. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in
Yaounde, Cameroon. J Infect Dis 183: 1828–1831.[ISI][Medline]

21. Cooper RA, Ferdig MT, Su XZ, Ursos LM, Mu J, Nomura T, Fujioka H, Fidock DA, Roepe PD, Wellems TE, 2002.
Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine
resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum. Mol Pharmacol 61: 35–42.
[Abstract/Free Full Text]

22. Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE,
Plowe CV, Coulibaly D, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257–263.
[Abstract/Free Full Text]

23. Kyosiimire-Lugemwa J, Nalunkuma-Kazibwe AJ, Mujuzi G, Mulindwa H, Talisuna A, Egwang TG, 2002. The Lys-76-Thr
mutation in PfCRT and chloroquine resistance in Plasmodium falciparum isolates from Uganda. Trans R Soc Trop Med Hyg
96: 91–95.[ISI][Medline]

24. Mayor AG, Gomez-Olive X, Aponte JJ, Casimiro S, Mabunda S, Dgedge M, Barreto A, Alonso PL, 2001. Prevalence of the
K76T mutation in the putative Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene and its relation to
chloroquine resistance in Mozambique. J Infect Dis 183: 1413–1416.[ISI][Medline]

25. Pillai DR, Labbe AC, Vanisaveth V, Hongvangthong B, Pomphida S, Inkathone S, Zhong K, Kain KC, 2001. Plasmodium
falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183: 789–
795.[ISI][Medline]

26. Su X-Z, Kirkman LA, Fujioka H, Wellems TE, 1997. Complex polymorphisms in an ~330 kDa protein are linked to
chloroquine-resistant P falciparum in Southeast Asia and Africa. Cell 91: 593–603.[ISI][Medline]

27. Vieira PP, das Gracas Alecrim M, da Silva LH, Gonzalez-Jimenez I, Zalis MG. 2001. Analysis of the PfCRT K76T mutation
in Plasmodium falciparum isolates from the Amazon region of Brazil. J Infect Dis 183: 1832–1833.[ISI][Medline]

28. Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Magill AJ, Su X-Z, 2002. Genetic diversity and chloroquine
selective sweeps in Plasmodium falciparum. Nature 418: 320–323.[ISI][Medline]

29. Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D, 2001. High-level chloroquine resistance in
Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene
pfcrt and the multidrug resistance gene pfmdr1. J Infect Dis 183: 1535–1538.[ISI][Medline]

30. Maguire JD, Susanti AI, Krisin, Sismadi P, Fryauff DJ, Baird JK, 2001. The T76 mutation in the pfcrt gene of Plasmodium
falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia. Ann Trop Med Parasitol 95: 559–572.[ISI]
[Medline]

31. Rieckmann KH, Campbell GH, Sax LJ, Mrema JE, 1978. Drug sensitivity of Plasmodium falciparum: an in vitro
microtechnique. Lancet 1: 22–23.[ISI][Medline]
 POLYMORPHISM OF THE PLASMODIUM FALCIPARUM MULTIDRUG RESISTANCE AND CHLOROQUINE RESISTANCE
TRANSPORTER GENES AND IN VITRO SUSCEPTIBILITY TO AMINOQUINOLINES IN ISOLATES FROM THE
PERUVIAN AMAZON

MARIA CECILIA HUAMAN, NORMA RONCAL, SHUSUKE NAKAZAWA, TON THAT AI LONG, LUCIA GERENA, CORALITH
GARCIA, LELY SOLARI, ALAN J. MAGILL, AND HIROJI KANBARA
Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan; Instituto de Medicina Tropical Alexander von Humboldt,
Universidad Peruana Cayetano Heredia, Lima, Peru; Department of Parasitology, Naval Medical Research Center Detachment,
Lima, Peru; Walter Reed Army Institute of Research, Silver Spring, Maryland

ABSTRACT
In vitro drug sensitivity to chloroquine (CQ), mefloquine (MQ) and quinine was investigated in 60 culture-adapted Plasmodium
falciparum isolates from malaria patients in Padrecocha, a village in the Amazonian Department of Loreto, Peru. All isolates showed
resistance to CQ, decreased susceptibility to quinine, and sensitivity to MQ. These isolates were examined for mutations in the P.
falciparum multidrug resistance 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes previously linked to CQ resistance.
The mutations N86Y and D1246Y, two of the five mutations commonly observed in the pfmdr1 gene of CQ-resistant clones, were not
found. The pfcrt mutation K76T, associated with CQ resistance, was identified in all the isolates tested. Sequence analysis of codons
72–76 in the pfcrt gene showed the haplotypes SVMNT and CVMNT.

INTRODUCTION

Malaria is increasingly a serious burden in most tropical countries and a major cause of death in children in sub-Saharan Africa. The
situation became more difficult since resistant Plasmodium falciparum strains appeared in late 1950s.1–3 In South America, the first P.
falciparum strains resistant to chloroquine (CQ) were reported in 1961 in Colombia4 and Brazil.5
Peru, the country with the second highest number of malaria cases in South America,6,7 used CQ extensively until 1998. In 1999, the
National Malaria Control Program of the National Institutes of Health of Peru8 reported 30–70% therapeutic failures to CQ.
Subsequently, the combination of sulfadoxine/pyrimethamine (S/P) was introduced for the treatment of uncomplicated P. falciparum
malaria. Single-dose primaquine was also given concurrently as a gametocytocidal agent.
Since CQ resistance was first described, great efforts have been made to understand the mechanism and, two relevant proteins,
Pgh19 and PfCRT,10 have been identified as candidates that participate in CQ resistance. The P. falciparum multidrug resistance gene
1 (pfmdr1) analog codes for Pgh1 and the P. falciparum chloroquine resistance transporter gene (pfcrt) codes for PfCRT. These
genes are located on chromosomes 5 and 7, respectively. Both proteins are localized in the food vacuole membrane and may
modulate CQ uptake and/ or pH regulation.11
It has been proposed that point mutations in the pfmdr1 gene producing amino acid changes at positions 86, 184, 1034, 1042, and
1246 are associated with CQ and quinine resistance, as well as increased levels of susceptibility to mefloquine (MQ).9,12 In other
studies, pfcrt mutations at codons 74, 75, 76, 220, 271, 326, 356, and 371, have been related to CQ resistance.11 Notably, mutation
K76T is consistently found in CQ-resistant strains11,13 and its contribution to CQ resistance has been recently elucidated by
transfection experiments.14 Results from experiments conducted with laboratory strains need to be corroborated by those obtained
from field isolate studies. Based on field studies, geographic variation in the parasite line due to regional differences has been
observed.11,15
There is little information in Peru about molecular characterization of P. falciparum strains. To determine the prevalence of CQ
resistance-associated markers, haplotype analysis of the pfcrt and pfmdr1 genes was performed in Peruvian P. falciparum isolates
from the Amazonian Department of Loreto.
MATERIALS AND METHODS

Study site. Padrecocha is a village of 1,400 inhabitants on the Nanay River, 5 km northwest of Iquitos, the departmental capital of
Loreto, Peru. An epidemiologic study16 was carried out in Padrecocha from August 1997 to July 1998, and of 4,046 blood smears
obtained, 36% were positive for malaria parasites. Plasmodium falciparum was found in 17% of all positive cases and P. vivax was
found in 83%. Due to widespread and frequent therapeutic failures of CQ for P. falciparum malaria in this area, the currently
recommended treatment is S/P, 25 mg and 1.25 mg/kg, respectively, and primaquine, 0.75 mg/kg administrated as a single dose.
Therapeutic failures with S/P are treated with quinine and tetracycline in adults or quinine and clindamycin in children. Plasmodium
vivax malaria is treated with a standard regimen of CQ for three days.
Isolates of P. falciparum. Plasmodium falciparum isolates were collected from 64 patients with uncomplicated acute P. falciparum
malaria in Padrecocha from March to May 1999. Patients enrolled in the therapeutic efficacy trial were asked to donate 5 mL of
venous blood. The trial was conducted with a modified version of the Pan American Health Organization template protocol for
conducting therapeutic efficacy trials in the Americas.17 Individual, written, informed consent was obtained from all participants. The
trial was conducted under the Walter Reed Army Institute of Research protocol No. 719 approved by the U. S. Army Surgeon General
Human Subjects Research Review Board and the Universidad Peruana Cayetano Heredia Institutional Review Board. Each sample
was collected into two microtubes and cryopreserved in liquid nitrogen as previously described18 in Iquitos, and later transported to the
Naval Medical Research Center Detachment Laboratory in Lima. One microtube of each sample was used for the drug resistance test
after being successfully culture adapted. The second tube was used for DNA extraction and gene analysis.
Laboratory strains. The CQ-sensitive P. falciparum strain FCR3 from The Gambia and the CQ-resistant strain K1 from Thailand
were used as controls for the detection of pfmdr1 polymorphism and direct sequencing analysis of pfcrt gene. These strains were
propagated in vitro in the Department of Protozoology, Institute of Tropical Medicine, Nagasaki University (Nagasaki, Japan).
Drug sensitivity testing. In vitro susceptibilities to antimalarials were examined by the inhibition test of 3H-labeled hypoxanthine
uptake in cultures of field-collected parasites, as previously described.19 Briefly, a microtube sample was thawed for culture adaptation
of 2–6 weeks. Sixty of the 64 collected samples successfully adapted to culture were tested for susceptibility to CQ diphosphate, MQ
hydrochloride, and quinine citrate. Drugs were dispensed in 25-µL aliquots of two-fold serial dilutions into 96-well, flat bottom
microplates to achieve the following final concentrations: CQ = CQ: 1,000 ng/mL (0.98 ng/mL), quinine = 1,000 ng/mL (0.98 ng/ mL),
and MQ = 250 ng/mL (0.24 ng/mL).
Two strains of P. falciparum were used as controls for the in vitro assays: strain D6, which is MQ resistant but otherwise drug
sensitive and strain W2, which is CQ and quinine resistant, but MQ sensitive.20,21 Both strains were obtained from Dr. Dennis Kyle
(Walter Reed Army Institute of Research, Washington DC).
The results of these assays are reported as the 50% inhibitory concentration (IC50) of the respective drug, that is, the concentration of
drug, usually in ng/mL, added to the culture that inhibits 50% of growth of the parasites.
In vitro CQ resistance can be defined as the ability of P. falciparum isolates to grow at a CQ concentration of 100 nM or 33 ng/mL in
culture.3,21 The cut-off value nicely separates CQ sensitivity, which is well below 33 ng/mL, and CQ resistance, which is above the cut-
off value. Clear, reproducible, and unambiguous results can be obtained with carefully controlled assay conditions that avoid pH
variations, variable serum activity, inaccurate gas conditions, inoculum effects, and other variations such as poorly growing parasites.
Extraction of DNA. Two hundred microliters each from 60 samples was subjected to DNA extraction using QIAamp DNA Blood kit
(Qiagen, Valencia, CA) to yield 200 µL of genomic DNA. The DNA was then eluted and stored in elution buffer, according to the
manufacturer’s instructions and used for a polymerase chain reaction (PCR) immediately or stored at –20°C.
Detection of pfmdr1 gene polymorphism. To determine the presence of sequence polymorphisms in pfmdr1, a PCR-restriction
fragment length polymorphism method was used. For this purpose, a nested PCR procedure (nest 1, nest 2) was carried out. The
region encompassing the polymorphic codons 86 and 184 was amplified using primers dr2 (5'-AGATGGTAACCTCAGTAT-3') and dr3
(5'-AGTCTTTTCTCCACAATA-3') and region encompassing the polymorphic codons 1034, 1042, and 1246 was obtained using
primers dr5 (5'-GAAATGTTTAAAGATCCAAG-3') and dr6 (5'-CAGCAAACTTACTAACAC-3') in nest 1. The design of primers was
based on the complete nucleotide sequence of the previously published GH2 strain (National Center for Biotechnology Information
Gene Bank, accession number S53996). Figure 1 shows the amplification strategy based on a previously reported protocol.22 Nest 1
amplification conditions were one cycle at 94°C for two minutes, an amplification of 35 cycles (94°C for 30 seconds, 45°C for one
minute, and 72°C for one minute), and a final extension at 72°C for five minutes. Two microliters of PCR products obtained in nest 1
were used for the second amplification, nest 2, by using primers A4, A2, 1034f, 1042r, 1246f, and dr6. The amplification conditions for
nest 2 were as previously reported and the products were subjected to enzyme digestion.22 This technique produces digestion
patterns corresponding to alternative polymorphisms following resolution by electrophoresis on 1–3% agarose gels (Nusieve 3:1; Bio-
Whittaker Molecular Applications, Rockland, ME). The reaction conditions for enzyme digestion were as previously described.22 The
PCR reagents were obtained from the TaKaRa Shuzo Co. (Kyoto, Japan). Restriction enzyme digestions were done with Apo I (New
England Biolabs, Inc., Beverly, MA) Dra I (Takara Shuzo Co.), Dde I (Sigma-Aldrich, Inc., St. Louis, MO), Vsp I (Gibco-BRL,
Gaithersburg, MD), and Eco RV (Takara Shuzo Co.) for the characterization of codons 86, 184, 1034, 1042, and 1246, respectively.

Amplification of the pfcrt gene. To amplify the pfcrt gene, a PCR was carried out using primers cr5 (5'-
TATAGATTATTTTCATTGTCTTCCACAT-3') and cr6 (5'-TTCCTTATAAAGTGT AATGCGATAG-3') in nest 1. For the second
amplification, nest 2, the previously reported primers 23402up and 24011dn15 were used to amplify the region encompassing the
polymorphic codons 72–76 and 97 in exon 2. Nest 1 amplification conditions were one cycle at 94°C for two minutes, an amplification
of 35 cycles (94°C for 30 seconds, 50°C for one minute, and 60°C for two minutes), and a final extension at 60°C for 10 minutes. Nest
2 amplification was carried out according to previously reported conditions.15
Direct DNA sequence analysis. The PCR amplification products were purified using a QIAquick PCR purification kit (Qiagen) and
sequenced directly on an ABI310 automated sequencer using the ABI PRISM Big Dye Terminator Cycle kit (Applied Biosystems,
Foster City, CA) following the manufacturer’s instructions.
Data and statistical analysis. Data for the in vitro drug sensitivity test were analyzed by non-linear regression. The IC50 of tested
isolates was compared with that of the D6 and W2 strains, and the mean IC50s swere compared using the Student’s t-test. SPSS
software for Windows (SPSS Inc., Chicago, IL) was used.

RESULTS

Susceptibility of P. falciparum isolates to CQ, MQ, and quinine. The in vitro susceptibility data for CQ, MQ, and quinine for 60
Peruvian isolates, and the profile of control strains D6 and W2 are shown in Table 1 . The IC50s to CQ of the isolates ranged from 29
to 35 ng/mL and were much higher than the one showed by the CQ-sensitive control strain, which ranged from 1 to 5 ng/mL. The
Peruvian isolates were at the lower level of IC50 values usually seen in CQ-resistant isolates. All isolates showed high IC50 values for
quinine, ranging from 45 to 56 ng/mL, consistent with decreased susceptibility. They also showed low IC50 values for MQ, which
ranged from 2.06 to 2.41 ng/mL, consistent with sensitivity to this drug.

TABLE 1
In vitro susceptibilities of the Peruvian isolates and control strains of Plasmodium falciparum to chloroquine (CQ), mefloquine (MQ),
and quinine*

Polymorphism of the pfmdr1 and pfcrt genes. Analysis of the pfmdr1 gene showed wild type codons at positions 86 (Asn) and
1246 (Asp). However, at codons 184, 1034, and 1042, the substitutions Y184F, S1034C, and N1042D were found in all samples. The
pfmdr1 polymorphisms at codons 86 and 184 of representative Peruvian P. falciparum isolates are shown in Figure 2 . The pfmdr1
haplotype for all 60 Peruvian isolates was NFCDD for positions 86, 184, 1034, 1042, and 1246, respectively.
FIGURE 2. Agarose gel electrophoresis of restriction enzyme-digested products used in the polymorphism analysis of the
Plasmodium falciparum multidrug resistance 1 gene in Peruvian isolates. A, Apo I digest for codon 86. Peruvian isolates show wild-
type Asn. B, Dra I digest for codon 184. Peruvian isolates show the Y184F mutation. U = undigested product; K1 = P. falciparum K1
strain; M = molecular size markers. Values along the gels are in basepairs.

Analysis of the pfcrt gene showed that the K76T substitution was present in all Peruvian P. falciparum isolates evaluated. The DNA
sequences at polymorphic codons 72, 74, 75, and 97 were also analyzed. At position 72, cysteine and serine (encoded by TCT, Stct)
were found. The deduced haplotypes of the pfcrt gene at positions 72–76 and 97 are shown in Table 2 .
TABLE 2
Plasmodium falciparum chloroquine resistance transporter gene mutations in isolates from Peru*

Genotyping of P. falciparum. Codons 72–76 of the Peruvian isolates were sequenced and the haplotypes SVMNT (30 of 60, 50%)
and CVMNT were found. CVMNT and SVMNT are haplotypes previously reported for laboratory strains from Ecuador and Brazil,
respectively,11 and in field samples from Peru.23 These two haplotypes showed the same in vitro susceptibility pattern to CQ (P =
0.21), MQ (P = 0.69), and quinine (P = 0.11). Figure 3 shows scatter plots of the data. No polyclonal infections were found in any of
the tested alleles.
FIGURE 3. In vitro susceptibilities to chloroquine, mefloquine, and quinine in Peruvian isolates of Plasmodium falciparum with the
deduced CVMNT and SVMNT haplotypes in the P. falciparum chloroquine resistance transporter (pfcrt) gene. 50% inhibitory
concentrations (IC50s) were compared using the Student’s t-test. Bars show the mean and 95% confidence intervals.

DISCUSSION

In this study, we report the prevalence of known genetic polymorphisms of the pfmdr1 and pfcrt genes and the in vitro drug
susceptibility profiles against the aminoquinoline-based antimalarial drugs in P. falciparum isolates from the Peruvian Amazon.
A total of 60 culture-adapted isolates were subjected to the drug sensitivity test for CQ, quinine, and MQ. All isolates were moderately
CQ resistant. This result was not unexpected since CQ resistance have been observed in Peru since 1992,24 and more than 30% of
the reported cases of malaria in 1998 showed therapeutic failure to CQ treatment.25 The fact that all isolates showed a narrow range
of IC50s against the three aminoquinoline-based drugs tested might reflect the presence of a uniform P. falciparum population in
Padreco-cha village. Therefore, imported clones rather than indigenous parasites, which had acquired drug resistance, might have
spread in the area. Conversely, we should consider that these isolates do not entirely represent the P. falciparum population because
they were once selected by a culture condition.
The mutation Y184F was identified in the pfmdr1 gene of all CQ-resistant Peruvian strains analyzed in this study. It had been
previously reported for CQ-resistant laboratory strains9 and field isolates.26 However, the mutation N86Y was not found in the same
CQ-resistant strains, in contrast to studies carried out in Malaysia,27 Indonesia,28 Guinea-Bissau,29 Nigeria,30 and sub-Saharan Africa,31
but in agreement with a study carried out in Brazil.26 Other clinical studies in Uganda,32 Laos,33 Cameroon,34 and southern Africa35
reported that the N86Y mutation in pfmdr1 was not predictive of treatment outcome. In addition to the mutations Y184F and N86Y,
other three-point mutations in pfmdr1 gene, previously related to CQ resistance, quinine resistance, and MQ sensitivity, were studied.
The point mutations S1034C and N1042D were identified in our isolates, but D1246Y was not detected. Reed and others12
investigated the role of Cys 1034, Asp 1042, and Tyr 1246 in aminoquinoline-based drug resistance by using transfection technology.
They reported that the three mutations play a role in resistance to quinine, as well as in the sensitivity to MQ, and suggested that Tyr
1246 is a pivotal player in MQ sensitivity. This suggestion is contradictory to the results with our MQ-sensitive isolates, in which such a
mutation was absent.
Previous studies evaluated pfmdr1 and pfcrt genes, and suggested that mutations in pfmdr1 may confer some advantage to the
parasite in the presence of CQ, increasing the level of CQ resistance.36,37 When mutations are present in both genes, IC50s to CQ are
higher than when only mutations in the pfcrt gene are found. In our study, two of five mutations studied in pfmdr1 were not present in
the Peruvian isolates. This may explain the low levels of CQ resistance observed in our isolates. The detection of only one pattern of
pfmdr1 mutations in this area again supports the idea that imported clones with the same drug-resistant characteristics might spread.
With regard to the pfcrt gene, all Peruvian isolates showed the CQ-resistant phenotype and the K76T mutation. Our results are in
agreement with those reported by Fidock and others,11 Djimde and others,36 and Vieira and others38 on in vitro susceptibility profiles. In
addition, previous reports suggested that this mutation is the major determinant for CQ resistance, but the clinical outcome might
involve other factors.32–34,37
Sequencing of the pfcrt gene identified two haplotypes, CVMNT and SVMNT, at codons 72–76. The SVMNT haplotype was
previously found in laboratory strains of P. falciparum from Brazil,15 and the CVMNT haplotype was related to Ecuadorian and
Colombian strains.15 Recently, Cortese and others23 studied drug resistance mutations in South American isolates, including 16
Peruvian samples, and identified these two haplotypes.
Aramburu and others6 speculated that three types of drug-resistant P. falciparum isolates converged in the Iquitos region of Peru.
These are S/P-resistant CQ-resistant parasites from Brazil, variably S/P-resistant CQ-resistant parasites from the Loreto region, and
S/P-sensitive CQ-sensitive parasites from coastal Peru. With regard to this hypothesis, our study suggests that the P. falciparum
population in Padrecocha village in Iquitos includes Brazilian strains with the SVMNT haplotype, which correlates well with CQ and
S/P resistance (Huaman MC and others, unpublished data), and Loretana strains with the CVMNT haplotype, which correlates with
CQ resistance and S/P variable resistance. This finding suggests that SVMNT and CVMNT haplotypes might be useful markers of
strain origin that would be complementary to merozoite surface protein-1 (MSP-1), MSP-2, glutamate-rich protein, and microsatellite
markers.
In conclusion, we suggest that the two types of P. falciparum strains with low-grade resistance to aminoquinolines were recently
introduced into Iquitos in the Peruvian Amazon and spread without further mutations.
.
REFERENCES

1. Payne D, 1987. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol Today 3: 241–246.[ISI]

2. Wongsrichanalai C. Pickard AL, Wernsdorfer WH, Meshnick SR. 2002. Epidemiology of drug-resistant malaria. Lancet Infect
Dis 2: 209–218.[ISI][Medline]

3. Wellems TE. Plowe CV. 2001. Chloroquine-resistant malaria. J Infect Dis 184: 770–776.[ISI][Medline]

4. Moore DV, Lanier JE, 1961. Observations on two Plasmodium falciparum infectious with abnormal response to chloroquine.
Am J Trop Med Hyg 10: 5–6.[ISI][Medline]
5. Rodriguez DP, 1961. Casos de malaria por Plasmodium falciparum resistentes ao tratamento pela chloroquina. Arch
Higiene Saude Publica 26: 231–235.

6. Aramburu J, Ramal C, Witzig R, 1999. Malaria reemergence in the Peruvian Amazon region. Emerg Infect Dis 5: 209–215.
[ISI][Medline]

7. WHO, 1999. World Health Report 1999. http://www.who.int/whr/1999/en/report.htm.

8. Instituto Nacional de Salud, 2000. National Policy of Antimalarials in Peru. Report of the National Program for Malaria
Control. Lima, Peru: MINSA.

9. Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cowman AF, 1990. Several alleles of the multidrug-
resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 345: 255–258.[ISI][Medline]

10. Wellems TE, Walker-Jonah A, Panton LJ, 1991. Genetic mapping of the chloroquine-resistance locus on Plasmodium
falciparum chromosome 7. Proc Natl Acad Sci USA 88: 3382–3386.[Abstract]

11. Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LMB, Singh Sidhu AB, Naude B, Deitsch
KW, Su XZ, Wootton JC, Roepe PD, Wellems TE, 2000. Mutations n the P. falciparum digestive vacuole transmembrane
protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6: 861–871.[ISI][Medline]

12. Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF, 2000. Pgh1 modulates sensitivity and resistance to multiple
antimalarials in Plasmodium falciparum. Nature 403: 906–909.[ISI][Medline]

13. Chen N, Russell B, Staley J, Kotecka B, Nasveld P, Cheng Q, 2001. Sequence polymorphisms in pfcrt are strongly
associated with chloroquine resistance in Plasmodium falciparum. J Infect Dis 183: 1543–1545.[Medline]

14. Singh Sidhu AB, Verdier-Pinard D, Fidock DA, 2002. Chloroquine Resistance in Plasmodium falciparum malaria parasites
conferred by pfcrt mutations. Science 298: 210–213.[Abstract/Free Full Text]

15. Mehlotra RK, Fujioka H, Roepe PD, Janeeh O, Ursos LMB, Jacobs-Lorena V, McNamara DT, Bockarie MJ, Kazura JW,
Kyle DE, Fidock DA, Zimmerman PA, 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype
in association with pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci 98: 12689–12694.
[Abstract/Free Full Text]

16. Roper MH, Carrion RS, Cava CG, Andersen EM, Aramburu JS, Calampa C, Hightower AW, Magill AJ, 2000. The
epidemiology of malaria in an epidemic area of the Peruvian amazon. Am J Trop Med Hyg 62: 247–256.[Abstract/Free
Full Text]

17. Pan American Health Organization, 1998. Evaluation of the Therapeutic Efficacy of Drugs for Treatment of Uncomplicated
Plasmodium falciparum Malaria in the Americas. OPS/HCP/ HCT/113/98 Washington, DC: Pan American Health
Organization.

18. Pavanand K, Permpanich B, Chuanak N, Sookto P, 1974. Preservation of Plasmodium falciparum-infected erythrocytes for
in vitro culture. J Parasitol 60: 537–539.[ISI][Medline]

19. Webster HK, Boudreau EF, Pavanand K, Yongvanitchit K, Pang LW, 1985. Antimalarial drug susceptibility testing of
Plasmodium falciparum in Thailand using a microdilution radioisotope method. Am J Trop Med Hyg 34: 228–235.[ISI]
[Medline]

20. Milhous WK, Gerena L, Kyle DE, Oduola AM, 1989. In vitro strategies for circumventing antimalarial drug resistance. Prog
Clin Biol Res 313: 61–72.[Medline]

21. Rathod PK, McErlean T, Lee PC, 1997. Variations in frequencies of drug resistance in Plasmodium falciparum. Proc Natl
Acad Sci USA 94: 9389–9393.[Abstract/Free Full Text]

22. Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 2000. The tyrosine-86 allele of the pfmdr 1
gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol
Biochem Parasitol 108: 13–23.[ISI][Medline]

23. Cortese JF, Caraballo A, Contreras CE, Plowe CV, 2002. Origin and Dissemination of Plasmodium falciparum drug-
resistance mutations in South America. J Infect Dis 189: 999–1006.

24. Castro Rivera, 1993. Estudio epidemiologico del brote de malaria por Plasmodium falciparum en el Valle Bigote, Piura en
setiembre de 1992. Tesis para optar el Grado de Medico - Cirujano. Peru. Lima, Peru: Universidad Nacional de Piura.

25. Instituto Nacional de Salud, 1999. Resistencia del Plasmodium falciparum a Medicamentos Antimalaricos en el Peru. Lima,
Peru: Informe Tecnico.
26. Zalis MG, Pang L, Silveira MS, Milhous Wilbur K, Wirth D, 1998. Characterization of Plasmodium falciparum isolated from
the Amazon region of Brazil: evidence for quinine resistance. Am J Trop Med Hyg 58: 630–637.[Abstract/Free Full Text]

27. Cox-Singh J, Singh B, Alias A, Abdullah MS, 1995. Assessment of the association between three point mutations in
chloroquine-resistance in vitro of Malaysian Plasmodium falciparum isolates. Trans R Soc Trop Med Hyg 89: 436–437.[ISI]
[Medline]

28. Gomez-Saladin E, Fryauff DJ, Taylor WRJ, Laksana BS, Susanti AI, Purnomo, Subianto B, Richie TL. 1999. Plasmodium
falciparum mdr1 mutations and in vivo chloroquine resistance in Indonesia. Am J Trop Med Hyg 61: 240–244.[Abstract/Free
Full Text]

29. Adagu IS, Dias F, Pinheiro L, Rombo L, do Rosario V, Warhurst DC, 1996. Guinea Bissau: association of chloroquine
resistance of Plasmodium falciparum with Tyr86 allele of multiple drug-resistance gene pfmdr1. Trans R Soc Trop Med Hyg
90: 90–91.[ISI][Medline]

30. Adagu IS, Warhurst DC, Carucci DJ, Duraisingh MT, 1995. Pfmdr1 mutations and chloroquine-resistance in Plasmodium
falciparum isolates from Zaria, Nigeria. Trans R Soc Trop Med Hyg 89: 132.[ISI]

31. Basco LK, Le Bras J, Rhoades Z, Wilson CM, 1995. Analysis of pfmdr1 and drug susceptibility in fresh isolates of
Plasmodium falciparum from Sub Saharan Africa. Mol Biochem Parasitol 74: 157–166.[ISI][Medline]

32. Dorsey G, Kamya MR, Singh A, Rosenthal PJ, 2001. Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr-1 genes
and clinical response to chloroquine in Kampala, Uganda. J Infect Dis 183: 1417–1420.[ISI][Medline]

33. Pillai DR, Labbe AC, Vanisaveth V, Hongvangthong B, Pomphida S, Inkathone S, Zhong K, Kain KC, 2001. Plasmodium
falciparum malaria in Laos: Chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183:
789–795.[ISI][Medline]

34. Basco LK, Ringwald P, 2001. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in
Yaounde, Cameroon. J Infect Dis 183: 1828–1831.[ISI][Medline]

35. McCutcheon KR, Freese JA, Frean JA, Sharp BL, Markus MB, 1999. Two point mutations in the multidrug-resistance gene
homologue of Plasmodium falciparum, pfmdr1, are not useful predictors of in-vivo or in-vitro chloroquine resistance in
southern Africa. Trans R Soc Trop Med Hyg 93: 300–302.[ISI][Medline]

36. Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE,
Plowe CV. 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257–263.
[Abstract/Free Full Text]

37. Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D, 2001. High-level chloroquine resistance in
Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene
pfcrt and the multidrug resistance gene pfmdr1. J Infect Dis 183: 1535–1538.[ISI][Medline]

38. Vieira P, Alecrim MdG, da Silva LHP, Gonzales-Jimenez I, Zalis MG. 2001. Analysis of the PfCRT K76T Mutation in
Plasmodium falciparum Isolates from the Amazon Region of Brazil. J Infect Dis 183: 1832–1833.[ISI][Medline]