Am. J. Trop. Med. Hyg., 70(4), 2004, pp.

395–397
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

IN VITRO EFFICACY OF ANTIMALARIAL DRUGS AGAINST PLASMODIUM VIVAX

ON THE WESTERN BORDER OF THAILAND
KESINEE CHOTIVANICH, RACHANEE UDOMSANGPETCH, WIRONGRONG CHIERAKUL, PAUL N. NEWTON,
RONATRAI RUANGVEERAYUTH, SASITHON PUKRITTAYAKAMEE, SORNCHAI LOOAREESUWAN, AND
NICHOLAS J. WHITE
Faculty of Tropical Medicine, and Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok. Thailand; Centre
for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, United Kingdom; Mae Sot Hospital, Tak, Thailand

Abstract. The susceptibility of 20 isolates of Plasmodium vivax on the Thailand-Myanmar border to seven antima-
larial drugs was evaluated using the schizont maturation inhibition technique. The geometric mean 50% inhibition
concentration (IC50) values were quinine ⳱ 308 ng/mL, amodiaquine ⳱ 14 ng/mL, chloroquine ⳱ 50 ng/mL, mefloquine
⳱ 127 ng/mL, sulfadoxine/pyrimethamine (80:1) ⳱ 800/10 ng/mL, pyrimethamine ⳱ 8 ng/mL, and artesunate ⳱ 0.5
ng/mL. Compared with P. falciparum in this area, P. vivax was more sensitive to chloroquine and artesunate, equally
sensitive to quinine, and more resistant to mefloquine.

INTRODUCTION quine were measured by a semi-quantitative dipstick tech-

Plamodium vivax is the major cause of human malaria in
parts of Central and South America and Asia. On the western
border of Thailand, the incidence of P. vivax has recently
been reported as 20 per 1,000 population per year, (which is
similar to that of P. falciparum1). Although severe complica-
tions of vivax malaria are rarely observed, P. vivax causes
multiple relapses and therefore considerable morbidity.
Vivax malaria in pregnancy is associated with low birth
weight.2,3 Choroquine has been the drug of choice for P. vivax
malaria for many years. In recent years, resistance to chloro-
quine in P. vivax has been demonstrated conclusively in vivo
in Papua New Guinea,4 different regions of Indonesia,5 and
more recently in central America.6 There have also been re-
ports from India7 and Myanmar.8 The susceptibility of P. vivax
to antimalarial drugs has not been monitored in vitro because
of difficulties in cultivating P. vivax. These difficulties are
related to differences in nutrient requirements compared with
P. falciparum; the conditions in standard malaria culture me-
dium induced premature rupture of the infected red blood
cells, and the limited provision of young red bloods cells or
reticulocytes limits P. vivax invasion. Plasmodium vivax in-
vades red blood cells only in the first 14 days after their emer-
gence from the bone marrow.9 In this study, the susceptibility
of P. vivax in vitro to different antimalrial drugs has been
assessed in short-term culture in an area where multi-drug
resistant P. falciparum is prevalent, and where several differ-
ent antimalarial drugs are available and used for treatment, to
set a baseline for P. vivax sensitivity in vitro.
MATERIALS AND METHODS
Study site. The studies were carried out in Mae Sot Hospi-
tal between 1996 and 2001 in Tak Province, NW Thailand.
This study was part of clinical studies reviewed and approved
by the Ethical and Scientific Review Committee, Ministry of
Public Health, Royal Government of Thailand.
Parasites. Two milliliters of blood were collected in hepa-
rinized tubes from patients attending the out-patient clinic or
admitted to Mae Sot Hospital. Thick and thin blood films
were prepared using standard procedures. Parasite species,
morphology, and parasitemia were assessed by microscopic
examination. Plasma concentrations of quinine and meflo-
nique.10 Only blood samples from patients with single-species
P. vivax malaria with synchronized ring-stage infected red
blood cells, parasitemia more than four parasitized red blood
cells per 1,000 red blood cells, and no antimalarial drug de-
tected in plasma (and no history of antimalarial drug treat-
ment) were chosen for this study.
Antimalarial drug sensitivity assay. Plasmodium vivax-
infected blood was centrifuged at 2,000 rpm at 4°C for five
minutes. After the plasma and buffy coat were discarded, the
packed red blood cells were washed three times in RPMI 1640
medium, and resuspended to make a 3% cell suspension in
the complete medium. A 50-␮L cell suspension was added to
triplicate wells of a predosed antimalarial microtiter plate.
The following drugs were assessed; quinine, amodiaquine,
chloroquine, mefloquine, sulfadoxine-pyrimethamine, and
pyrimethamine (World Health Organization, Manila, The
Philippines). The concentration (two-fold dilutions) ranges
for each drug used on the microtiter plates are shown in Table
1. Artesunate was prepared in duplicate wells in 96-well
plates as described previously11 200 ␮L of red blood cell sus-
pension was added into each well. Each drug concentration
was tested in duplicate. After adding the erythrocytes, the lid
was placed over the plate and the plate was then shaken
gently to dissolve the drug. The samples were incubated at
37°C in an atmosphere of 5% CO2 for 40−44 hours depending
on the stage of the parasite before culturing. At the end of the
incubation, thick and thin blood films were prepared from the
samples in each well. Wells without drugs were included as
controls.
Evaluation efficacy of antimalarial drugs. Thick and thin
blood films were fixed with methanol, and stained with Field’s
stain, and examined under the microscope. The number of
schizonts containing more than eight nuclei was counted per
3,000 red blood cells. The percentage of schizonts in the drug
wells was compared with the number of schizonts counted in
control samples.
Data analysis. Results of parasite schizogony (for 3,000 red
blood cells) after 40−48 hours culture at each drug concen-
tration were fitted to a sigmoid curve by using WinNonlin™
computer software version 3.1 (Pharsight Corporation,
Mountain View, CA) to determine the 50% inhibitory con-
centration (IC50) for schizont development. Correlations
were assessed by the method of Spearman.

395
396 CHOTIVANICH AND OTHERS

TABLE 1 earlier studies from the same region on P. falciparum in which

Drug concentration range tested schizont maturation was used,13 and with contemporary stud-
ies using 3H-hypoxanthine uptake inhibition.11
Drug Drug concentration

Quinine 4–256 pmol/well

Amodiaquine 0.3–16 pmol/well
Chloroquine 1–64 pmol/well
Mefloquine 2–128 pmol/well
Sulfadoxine/pyrimethamine (80:1) 10–10,000 pmol/well
Pyrimethamine 0.01–10,000 pmol/well
Artesunate 0.5–33.5 ng/mL/well
RESULTS
Twenty isolates were obtained. Growth of P. vivax in the
absence of antimalarials drugs was observed in all control
wells. The parasites developed from ring stage to mature
schizonts containing ⱖ 8 nuclei within a mean ± SD 40 ± 3
hours after cultivation. All 20 isolates used in the experiment
developed to complete mature schizonts and could be evalu-
ated for drug responsiveness. Tests were run in parallel for
the seven antimalarial drugs. Parasitemia before testing var-
ied between 0.4% and 1.9% (geometric mean ± 95% confi-
dence interval [CI] ⳱ 0.5 ± 0.2%). After 40 hours of incuba-
tion, the number of schizonts in the control wells of the seven
parallel series varied between 5 and 19 per 3,000 red blood
cells (geometric mean ± 95% CI ⳱ 8.6 ± 0.2). The overall
coefficient of variation for the counting of the individual iso-
lates’ control well, was 8%, indicating reproducibility of as-
sessment of schizont maturation.
The drug concentrations that gave 50% inhibition of schiz-
ont maturation (IC50) were derived from the sigmoid plots
and are summarized in Table 2. For most (19 of 20) of the
isolates, complete inhibition of schizont maturation occurred
in the well containing 64 pmol/well of quinine. This concen-
tration is considered to represent susceptibility to quinine for
P. falciparum.12 Complete inhibition of schizont maturation
(all isolates) occurred in the well containing 16 pmol of chlo-
roquine/well. For mefloquine, complete inhibition of schizont
maturation was achieved for all isolates in the well containing
128 pmol/well. Complete inhibition of schizont maturation
occurred in the well containing 8 pmol/well for amodiaquine
(18 isolates), 10,000 pmol/well for sulfadoxine-pyrimethamine
(18 isolates) and pyrimethamine (all isolates), and 8.4 ng/ml
for artesunate (all isolates). No significant correlations were
found between the susceptibilities to the different antimalari-
als. As shown in Table 2, these data were compared with
DISCUSSION
The susceptibility to antimalarial drugs of P. vivax was as-
sessed by a rapid, simple, and readily field-adapted method.
This method is similar to that used for P. falciparum, although
many laboratories now use the radioisotopic hypoxanthine
incorporation assay to assess drug susceptibility in this para-
site. The two methods give similar results. When compared
with P. falciparum isolates from the same area (assessed by
both the hypoxanthine incorporation assay and earlier studies
from the same area using schizont maturation assessment),
P. vivax was as susceptible to quinine, more sensitive to chlo-
roquine, and more resistant to mefloquine. The P. vivax IC50
of artesunate was approximately three times lower than that
of P. falciparum, suggesting that artesunate is a potential
treatment for chloroquine-resistant P. vivax malaria. This
confirms in vivo observations; P. vivax is highly susceptible to
the artemisinin derivatives.14 The IC50 values of sulfadoxine-
pyrimethamine and pyrimethamine from this study could not
be compared with assessments of susceptibility in P. falci-
parum because of the differences in methodology and the
conditions in the culture medium. Folic acid is a direct com-
petitive antagonist of antifolate activity15 and p-aminobenzoic
acid (PABA) is a direct competitor for sulfonamides.16
Therefore the susceptibility of P. falciparum to sulfadoxine-
pyrimethamine and pyrimethamine are usually conducted
in culture medium deficient in folic acid. We have found that
P. vivax needs considerably more folic acid for complete
schizont maturation than P. falciparum and will not grow in
folate-deficient media. Thus, the IC50 values of sulfadoxine-
pyrimethamine and pyrimethamine in this study might not
reflect in vivo susceptibility, but could be useful for sequential
monitoring of the evolution of antifolate resistance.
In Thailand, P. vivax remains sensitive to chloroquine
in vitro and in vivo. The low IC50s obtained in this study which
in P. falciparum would correspond to chloroquine sensitiv-
ity17 confirm extensive clinical series indicating that there is
no significant resistance in this region. This contrasts with
P. falciparum, which is highly chloroquine resistant in this
area. The IC50 values observed for chloroquine are consistent
with previous reports.18 Whether the relatively high IC50
value for mefloquine reflect: acquired resistance, as in P. fal-
ciparum in this region, or constitutionally reduced susceptibil-

TABLE 2
Susceptibility of Plasmodium vivax on the western border of Thailand to antimalarial drugs*

Plasmodium vivax Plasmodium falciparum

Geometric mean Brockman and others11 Childs and others13

Drug IC50 (ng/mL) 95% CI IC50 (ng/mL) (3H-hypoxanthine) IC50 (ng/mL) (Schizont maturation)

Quinine 308 97–1,082 370 81

Amodiaquine 14 7–29
Chloroquine 50 38–90 149 70
Mefloquine 127 121–272 27 3†
S/P (80:1) 10 463–4,780
Pyrimethamine 8 0.1–483 4,083†
Artesunate 0.5 0.4–0.7 1.6
* IC50 ⳱ 50% inhibitory concentration; CI ⳱ confidence interval; S/P ⳱ sulfadoxine/pyrimethamine.
† These isolates were assessed after the development of high-level pyrimethamine resistance but before the advent of significant mefloquine resistance.
 ANTIMALARIAL DRUG SUSCEPTIBILITY OF P. VIVAX
ity will require assessment in other areas where mefloquine is
not used. These preliminary data suggest that there are simi-
lar in vitro “break-points” for antimalarial drug resistance in
the two malaria species. These results provide a current base
line of sensitivity of P. vivax to antimalarial drugs. The anti-
malarial drug susceptibility of P. vivax needs to be monitored
continuously to anticipate the emergence of resistance.
Received January 21, 2003. Accepted for publication September 17,
2003.
Acknowledgments: We thank the staff and nurses of Mae Sot Hos-
pital for their help and Dr. Jetsumon Prachumsri for her valuable
comments and suggestions.
Financial support: This work was a part of the Wellcome Trust-
Mahidol University Oxford Tropical Medicine Research Programme
funded by the Wellcome Trust of Great Britain.
Authors’ addresses: Kesinee Chotivanich, Wirongrong Chierakul,
Sasithon Pukrittayakamee, and Sornchai Looareesuwan, Faculty of
Tropical Medicine, Mahidol University, 420/6 Rajvithi Road. Bangkok,
Thailand. Rachanee Udomsangpetch, Department of Pathobiology,
Faculty of Science, Mahidol University. Bangkok, Thailand. Ronatrai
Ruangveerayuth, Mae Sot Hospital, Tak, Thailand. Paul N. Newton
and Nicholas J White, Faculty of Tropical Medicine, Mahidol Uni-
versity, 420/6 Rajvithi Road, Bangkok 10400, Thailand, and Centre
for Clinical Vaccinology and Tropical Medicine, Churchill Hospital,
Oxford OX3 7LJ, United Kingdom, Correspondence: Prof. N. J.
White, Telephone: 66-2-354-9172, Fax: 66-2-351-9169, E-mail:
fnnjw@diamond.mahidol.ac.th.
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