TRANSACTIONS

OF THE ROYAL

SOCIETY

OF TROPICAL

MEDICINE

AND HYGIENE

(2001) 95,325-329

Resistance of Plasmodium
falciparum to antimalarial
drugs in a highly
area of southern Viet Nam: a study in viva and in vitro

endemic

Nguyen Mai Huong’,
Sean Hewitt*,
Timothy
M. E. Davis2’3, Le Due Dao’, Tran Quoc Toan’,
‘Nutianal
Tran Bach Kim’, Nguyen Thi Hanh’, Vo Nhu Phuong’ , Doan Hanh Nhan’ and Le Dinh Gong’
Institute of Malariology,
Parasitology
and Entomology, Luong The Virzh Road, Hanoi, Viet Nam; ‘Vie, Nam-Australia
Malaria Control Project, Ministy

of Health, 138A Giang Vo, Hanoi, Viet Nam; 3 University of Western Australia, Department
of Medicine, Fremantle Hospital, Fremantle, Australia

Abstract
To assess the antimalarial sensitivity of Plasmodium falciparum in vivo and in vitro in a highly endemic area of
southern Viet Nam. a field studv was conducted (in 1999) at a rubber plantation in Binh Phuoc Province
north of Ho Chi r&h City. F&y patients were ireated with either a;esunate (4 mg/kg on day 0, then
2 mg/kg on day 1 to 4) or mefloquine (10 mg/kg at 0 h, then 5 mg/kg at 6 h), and their progress was
followed for 28 days under standard WHO protocols. Blood spots were taken at baseline from all patients, as
well as from those who redeveloped parasitaemia during follow-up, for polymerase chain reaction (PCR)
determination of parasite genotypes to assist differentiation of re-infection from recrudescence. Both
treatments cleared parasites within 5 days, Ofthe 25 mefloquine-treated patients, 2 (8%) re-presented with
probable re-infections. For artesunate, 4 patients (16%) had re-infections and 5 (20%) had recrudescences.
Sensitivity tests in vitro of pre-treatment I? fulciparum isolates showed geometric mean IC,, values of 29,38,
209 and 15 nmol/L for chloroquine (n = 32), mefloquine (n = 33), quinine (n = 31) and artemisinin
(n = 3 1), respectively. There were significant correlations between IC+ for artemisinin and mefloquine
(r = 0.72, P = 0.004), and chloroquine and quinine (r = 0.44, P = 0.05). These data show that, although
mefloquine has been used for 10 years in Binh Phuoc Province, it remains fully effective, perhaps because an
artemisinin derivative is commonly given at the same time. The recrudescence rate for artesunate is similar
to those reported in other epidemiological contexts. The present in-vitro data imply that quinine remains
effective and that reduced drug pressure has been associated with increased sensitivity of local strains of l?
falciparum
to chloroquine. Although from one hyperendemic area, these results may have implications for
antimalarial prophylaxis and treatment strategies for residents and travellers to southern Viet Nam.
malaria, Plasmodium falciparum,
artesunate, artemisinin, Viet Nam

Keywords:

drug sensitivity, in viva, in vitro, chloroquine, mefloquine, quinine,


Introduction
The threat of resistance of Plasmodium falciparum to
available antimalarial drugs remains of great concern,
especially in south-east Asian countries such as Viet
Nam. Although formal reports in the literature from
Indochina have been understandably few, clinical evidence of chloroquine-resistant
I? falciparum
was first
found in southern Viet Nam in 1961 and similar reports
from the central provinces of Quang Binh and Nghe An
followed 6 years later (PHAN, 1998). As a result, the
National Institute of Malariology, Parasitology and Entomology (NIMPE) in Hanoi established routine monitoring of drug resistance in I? falciparum in 1968. The
standard 7-day test recommended by the World Health
Organization (WHO) was used initially. The extended
28-day in-vivo test was introduced in 1980 together with
the WHO macro and micro in-vitro tests.
Drug-resistant I? falciparum is now widespread, particularlv in central and southern Viet Nam. Chloroauine
resistance in vivo is currently thought to range from*30%
to 85% while sulfadoxine-pyrimethamine
resistance is
encountered in 30-80% of patients (N. D. Sy, personal
communication). In response to the need for alternative
treatments, mefloquine and artemisinin derivatives were
introduced in 1990. Mefloquine use was limited initially
to combination therapy with artemisinin derivatives in
drug-resistant cases. However, mefloquine-artemisinin
combinations have had wider annlication since 1995 and
the use of chloroquine, sulfadokine-pyrimethamine
and

quinine has declined.
Although an attenuated in-vivo response to artemisinin has been described (LUXEMBERGER
et al., 1998),
evidence from Thailand suggests that mefloquine and
artemisinin may be murually protective when used in
combination (NOSTEN
et al., 1998; WHITE,
1998).
In
Address for correspondence: Professor T. M. E. Davis, University ofwestern Australia, Department of Medicine, Fremantle Hospital, P.O. Box 480, Fremantle, Western Australia 6959,
Australia; phone +618 9431 3229, fax +618 9431 2977, e-mail


the light of these reports, and given the history of drug use
and parasite resistance in Viet Nam, we aimed to establish the sensitivity of I? fulciparum to (i) artesunate and
mefloquine in vivo and in vitro, and (ii) chloroquine and
quinine in vitro in an endemic rural area in the south of
the country.
Methods
Study site

The study was approved by the Ethics and Scientific
Committee of NIMPE and nerformed at Phu Riena
rubber plantation, Phuoc Ling district, Binh Phuo:
province. Phuoc Long district is in a highly endemic area
in southern Viet Nam well known for multidrug-resistant
I? fulciparum.
Malaria transmission occurs all year
round, with peaks reported between May and June and
betweenNovember

and December. Between August and
October 1998, cross-sectional surveys revealed a-malaria
orevalence of23% 170% I? falciaarum. 27% I? vivax and
3% mixed infectiois). In &e early 1<9Os, 65% of cases
were thought to be resistant to chloroquine and 70% to
sulfadoxine-pyrimethamine,
both at the RI1 or RI11 level
(N. D. Sy, personal communication).
In-vivo

study

To assess the sensitivity in viva of I? falciparum
to
artesunate and mefloquine, 50 patients were recruited to
a randomized, parallgl, single-blind study using the 28day test (WHO, 1973). Suspected malaria cases seen at
Phu Rieng plantation between July and November 1999
were examined and their details recorded. Thick and thin
blood smears were treated with Giemsa stain and
checked by a skilled microscopist. Patients were invited
to take part in the study if they (i) gave informed consent
to study procedures, (ii) were aged between 4 and 65
years, and (iii) had uncomplicated falciparum malaria
with a parasite density of >lOOO/pL
whole blood.
Exclusion criteria included (i) pregnancy, (ii) concomitant illness, and (iii) prior antimalarial therapy, specifically quinine or an artemisinin drug within the previous 7
days, a 4-aminoquinoline within the previous 14 days, or


326


NGUYEN MA1 HUONG ETA,!,.

pyrimethamine
and/or sulphonamide within the previous 28 days. Dill-Glazko
urine tests (reagents preDared bv NIMPE) were nerformed to confirm that all
recruits had taken neither chloroquine nor sulfadoxinepyrimethamine.
All patients were admitted to hospital and duplicate
thick and thin blood films prepared. Twenty-five patients
were randomized to receive 5 days of artesunate (50 mg
salt, National Pharmaceutical Company No. 1, Hanoi,
Viet Nam), consisting of 4 mg/kg single dose on day 0
and then 2 mg/kg morning doses on days 1 to 4 inclusive.
A further 25 patients were randomized to a l-day course
of MephaquineTM (250 mg base, Mepha, Switzerland),
10 mg/kg at 0 h followed by 5 mg/kg at 6 h. This group
received antiemetic (atropine 0.1 mg/kg bodyweight)
and antipyretic
(paracetamol 10 mg/kg) medication
30 min before treatment was given. All treatments were
supervised and patients were observed for at least 60 min
post administration. Any patients vomiting during this
period were retreated but excluded from the study.
Oral temperatures were measured daily. As well as
those scheduled on davs 3 and 7. additional thick and
thin blood films were taken daily until parasite clearance
was confirmed by 2 consecutive negative results (no
asexual forms in 100 fields of view at X 1000 magnification). Patients were kept in hospital for at least 7 days and
were discharged when asymptomatic and aparasitaemic.
All were asked to return for follow-up on days 14,2 1 and

28 or if they became symptomatic. A blood film was
taken and a clinical assessment (including oral temperature) was performed on each of these occasions. Those
who failed to attend were contacted by local health
workers in an attempt to provide as complete a followup as possible.
A sensitive (S) response was recorded if no asexual
stages were found on day 7 and parasites had not
reappeared by day 28. If asexual parasites disappeared
by day 7 but reappeared before day 28 the isolate was
considered to have probable RI resistance. If the asexual
parasitaemia had dropped by at least 75% at 48 h but not
cleared, and if parasites were still present on day 7, the
parasites were considered resistant at the RI1 level. If the
asexual parasitaemia had dropped by less than 75% at
48 h and the patient remained slide-positive on day 7, the
parasites were considered resistant at the RI11 level.
Cytogenetic studies were carried out to distinguish
recrudescence from re-infection. Parasite DNA was
extracted from Whatman filter-paper using a modification of the method described bv KAIN & LANAR C199 1).
The blood-soaked portion of the papers was cut into fine
strips, placed in 1*5-mL polyethylene tubes with sodium
dodecyl sulphate (0.5% final concentration) and proteinase K (500 pg/mL final concentration) and incubated
at 37°C for 3 h. The DNA was then extracted with
phenol, precipitated with alcohol and amplified using
nested polymerase chain reaction (PCR). A set of
primers was chosen to identify 16 distinct association
types. These consisted of merozoite surface protein 1

(MSPl, block 2) allelic families (MAD20,
Kl and
R033), MSP2 allelic families (FC27 and Indochina),

and glutamate-rich protein (GLURP). PCR products
were analysed by gel electrophoresis.
In-vitro test
The WHO standard Mark III micro-test was applied
for in-vitro assessment of the drug sensitivity of parasites
from the patients recruited to the in-vivo study. Patients
with an asexual parasitaemia between 1000 and 80 000
parasites/pL were considered suitable for testing. In all,
parasite cultures from 33 patients were studied. These
cultures were tested against artemisinin, mefloquine,
chloroquine and quinine. The artemisinin plates used
were produced by NIMPE under guidance from WHO.
All others were supplied directly by WHO. For mefloquine, chloroquine, and quinine, parasites were considered sensitive if complete inhibition
of schizonts
occurred at 3200,800 and 2560 nmol/L or less, respectively. There is currently no sensitivity threshold set for
artemisinin.
Data analysis
Drug concentrations inhibiting parasite growth by
50% (ICsOs) were calculated using nonlinear regression
based on software developed by WERNSDORFER &
WERNSDORFER (1995) and are reported as geometric
means and 95% confidence intervals. Two sample comparisons were by Student’s t-test or, in the case of nonnormally distributed or discontinuous data, by Wilcoxon-Mann-Whitney
tests. The x2 test was used to assess
differences in proportions between groups. Associations
between variables were assessed using Pearson’s product-moment correlation co-efficient. A 2-tailed level of
significance was used throughout.
Results
Details of the patients are summarized in Table 1.
There were no significant differences in age, sex distribution, bodyweight or admission oral temperature
between the 2 groups (P>O.5). Although the day-0

median parasite density in the artesunate group was
significantly
greater than in the mefloquine group
(P = 0,024), the median parasite clearance time was
shorter (P = 0,004). Parasite clearance curves are shown
in the Figure. All patients were afebrile by day 2. The
majority of patients (85%) in the mefloquine group
reported
suffering side-effects including
dizziness
(80%), headache (64%), nausea (32%), vomiting
(16%), tremor (5%) and abdominal pain (5%). Symptoms were usually mild and patients recovered without
medical intervention. No side-effects were reported in
the artesunate group.
Data from in-vitro tests are summarized in Table 2.
Sensitivity in vitro to mefloquine was 100% amongst the
isolates tested, with complete inhibition of schizont
formation
occurring
in
the
wells
containing
320 nmol/L (the WHO discriminating concentration).

Table 1. Details of the Vietnamese malaria patients at the time of admission
to the study (July-November
1999), classified by allocated treatment
Characteristic


Mefloquine

Artesunate

Number of patients
Age (yea&
Sex (% males)
Bodyweight (kg)
Oral temperature (“C)
Parasitaemia (/@J
Parasite clearance time (days)
Fever clearance time (days)

25
26.5 + 13.3

26.2:

40.3 g-62)
38.8 It 0.8
7400 (1040-114000)
3 (l-5)
1 (O-2)

13.5

43.9 (qo5-68)
38.8 f 0.7
18 200 (1560-75 300)*
2 (l-4)*

1 (l-2)

Data are mean f SD or median (range). *P < 0.05 in comparison with the mefloquine group.


PLASMODIUM

FALCIPARUM

327

DRUG RESISTANCE IN VIET NAM

120000

.m
E
s 60000
.r
m"
40000
z

16

18

20

22


24

26

28

20000

0
4

3

2

1

Days after admission
Figure. Median (circles) and range (vertical bars) for I? fulciparum parasite clearance curves for artesunate (0) and mefloquine (0). The
upper right insert shows the patients who redeveloped parasitaemia during follow-up: mefloquine reinfection (grey bars), artesunate
reinfection (white bars) and artesunate recrudescence (black bars).

concentrations
for the four antimalarial
drugs evaluated
Table 2. Inhibitory
during in-vitro testing of Vietnamese
l? falciparum
isolates (1999)

Concentration
Dw
Chloroquine
Mefloquine
Quinine
Artemisinin

n
32
33
31
31

IC,,

IC,,
29
38
209
15.0

(22-39)
(28-50)
(155-282)
(9.4-24.1)

(nmol/L)

110
80

628
84.0

(72-169)
(53-121)
(406-973)
(43.4-162.5)

G9
323
148
1540
341

(170-612)
(79-276)
(805-2944)
(130-892)

Values are geometric means (95% confidence intervals in parentheses).

Among the 32 isolates tested successfully against chloroquine in vitro, 5 (16%) were resistant (discriminating
concentration 80 nmol/L). All 31 isolates tested against
quinine and artemisinin were sensitive to quinine (discriminating concentration 2560 nmol/L) and showed
complete schizont inhibition at artemisinin concentrations >300 nmol/L. Further analysis of the in-vitro
results for individual isolates revealed significant correlations between I&s
for artemisinin and mefloquine
(r = 0.72, P = 0.004), and chloroquine and quinine
(r = 0.44, P = 0.05).
Parasitaemia re-appeared in 2 patients from the mefloquine group (8%) and in 9 patients from the artesunate group (36%) between 14 and 28 days (P = 0.03).

Cytogenetic studies indicated that both the mefloquinetreated patients had re-infections while, in the artesunate
group, 4 patients (16%) had re-infections and 5 (20%)
recrudescences (see Figure). Cytogenetic studies that
were done on 49 of the 50 patients at the time of
admission to the study using the full primer set showed
that no 2 patients had the same PCR profile. It is very
unlikely, therefore, that the patients whose PCR results
were interpreted as recrudescences were, in reality, reinfected with the same strain. No patients exhibited
resistance at the RI1 or RI11 level. All 11 patients who
redeveloped a parasitaemia during follow-up were retreated with the alternative drug regimen and prompt
parasite clearance was observed in each case.
Of the 5 day-0 isolates taken from patients subsequently exhibiting recrudescence after artesunate therapy, 1 failed to grow in vitro, 2 displayed complete
schizont inhibition at 30 nmol/L artemisinin and 2 at

100 nmol/L artemisinin. One of these 2 latter isolates
also demonstrated relatively high tolerance to mefloquine with complete inhibition of schizont formation
occurring only in the well containing 320 nmol/L. In 2
patients who did not recrudesce after artesunate, inhibition of schizont formation occurred only at 1000 nmol/L
artemisinin.
Discussion
Our in-vivo data, supported by PCR analysis, provide
evidence that mefloquine remains a fully effective antimalarial drug despite being in use for almost a decade in
the endemic area of southern Viet Nam in which the
study was performed. The reason may be that mefloquine has been protected by the concomitant use of an
artemisinin derivative during this time. Artesunate
cleared parasites more rapidly than mefloquine but was
associated with recrudescence in 20% of patients as
assessed using PCR in paired blood spots. Nevertheless,
this recrudescence rate is similar to that reported by other
investigators who have also used a 5-day artesunate

regimen (BARRADELL & FI~TON, 1995). These in-vivo
findings are supported by the present in-vitro data.
Mefloquine
sensitivity was 100% based on current
WHO criteria. Isolates taken from the artesunate-treated
patients showed complete schizont inhibition in the
presence of artemisinin concentrations at or below
1 pmol/L. Our in-vitro data also provide evidence that
quinine is still an effective first-line agent against P.
falciparum
in Viet Nam, and suggest that reduced drug
pressure has had a significant positive impact on parasite
sensitivity to chloroquine.


328

NGUYENMAIHUONGETAL.

In Viet Nam, antimalarials are the only drugs supplied
by the health services to all groups free of charge. There
has, therefore, been the potential for overuse. Antimalarial treatment may be given to patients with fever
irrespective of other symptoms or given out in exchange
for blood smears during mass surveys. These practices
would favour the development of parasite resistance. The
dispensing of antimalarial drugs is, however, increasingly
restricted, and in many areas they are now provided only
for the treatment of clinically diagnosed malaria or as
single dose ‘prophylactic treatment’ for non-immune
forest workers.

Mefloquine has, however, been a special case. From
1984 to 1990, mefloquine was in widespread and often
indiscriminate use along the western border of Thailand.
As a result its antimalarial efficacy fell dramatically.
LOOAREESUWAN et al. (1992) reported that the cure rate
for a 15-mg/kg dose decreased from 98% in 1983-86 to
7 1% in 1930.IMefloquine
was first introduced to Viet
Nam in 1990 but, based on the reports from Thailand
and because of its relatively high cost, its use was limited
to combination therapy with artemisinin derivatives for
patients from areas with drug-resistant parasites. Only
over the past 4-5 years has such combination therapy
become more commonplace. Despite this trend, there
does not appear to have-been a decline in the efficacy of
either drug. In Thailand the introduction of artesunate in
1994 for use in combination with mefloquine prevented
further reductions in the efficacy of mefloquine (NOSTEN et al., 1998). This might also explain the 100% cure
rate observed in the present study, which is comparable
to that when mefloquine was first introduced to Viet
Nam as well as to rates recorded in 1993 and 1998 (N. D.
Sy & D. X. Huong, unpublished observations). Furthermore, sensitivity in vitro to mefloquine in the present
study was 100% and the geometric mean I&, was
38 nmol/L. These results were similar to those from
previous’ unpublished studies carried out in a variety of
locations in Viet Nam since 1986 in which 392%
sensitivity and an IC,, G83 nmol/L were found (V. T.
Tuyet, N. T. Tien, T. T. Tinh, N. V. Thanh, unpublished observations).
Despite the fact that the admission parasitaemia was
significantly higher in the artesunate than the mefloquine

group, parasite clearance occurred earlier in the former
group. There were no RI1 or RI11 cases, but a 5-day
artesunate regimen was associated with a recrudescence
rate of 20%. In many parts of Viet Nam, artemisinin and
artesunate have been used as a first-line treatment for
falciparum malaria for nearly 10 years. Up until now

Table 3. A summary
showing fluctuations

of unpublished
in recrudescence

studies carried
rates (%R)

there have been no documented reports of resistance in
viva but high recrudescence rates were reported from the
outset (see Table 3; NGUYEN, 1993). Longer regimens
give better cure rates (BUNNAG et al.. 1991) but patient
compliance falls off rapidly after the symptoms of malaria
subside. These considerations also argue for the combination of an artemisinin drug such as artesunate with a
second longer half-life drug such as mefloquine. The
geometric mean IC,, of 15.0 nmol/L for artemisinin
observed during this study was very similar to that of
14.5 nmol/L
from a study conducted at a nearby
hospital (15 km distant) in 1995 (N. V. Huong, unpublished report). However, the short shelf-life for artemisinin plates has caused problems in the past (F&E et al.,
1999). They must be kept at or below 4°C during
transport and storage, and should be used within 3

months. While refrigeration and storage during the
present study were optimized, the results of previous
similar in-vitro studies of artemisinin in Viet Nam may
not be valid. In view of the range of sensitivities we
encountered, especially in recrudescent cases, there is a
definite need for in-vitro surveillance of artemisinin
drugs to be maintained on a regular basis in areas in
which they are used.
Chloroquine remains first-line treatment for l? vivax
in Viet Nam, but in highly endemic southern and central
regions it has not officially been used to treat falciparum
malaria for more than 10 years. Among the 32 isolates we
tested against chloroquine in vitro, 84% were sensitive. A
comparison of these results with those from previous
unpublished studies in the area confirms that sensitivity
in vitro has risen dramatically, presumably owing to
reduction in drug pressure. In 1986 V. T. Tuyet reported
that 83% of 496 isolates he tested were resistant with an
IC,, of 900 nmol/L. In 1998 N. V. Thanh reported that
17% of 30 isolates tested were resistant with an IC,, of
359 nmol/L. Tuyet’s study was carried out in a hospital
and some of the patients recruited are likely to have had a
history of treatment failure. This sample would, therefore, not have been a representative one. Nevertheless,
the magnitude of the difference between the results from
1986 and the 1990s suggests that resistance was very
much greater then than now.
Consistent with the results of a recent large-scale
comparative trial in which quinine was as effective as
artemether in southern Vietnamese patients with severe
malaria (HIEN et al, 1996), all 3 1 isolates investigated in

the present study were sensitive to quinine in vitro. This
result is also similar to those from a number of recent
unpublished studies conducted in Viet Nam. In 1998 N.

out in Phu Rieng Commune,

Date

Drug

Regimen

1990
1991 Mar-May
199 1 May- Jun
199 1 Sep-Nov
1992 Ott-Dee
1993 Ott-Dee
1993 Mar-May
1994 Ott-Dee
1995 May-Jun
1995 Aug-Ott
1996 Ott-Dee

Artemisinin
Artesunate (C)
Artemisinin
Artemisinin
Artemisinin
Artemisinin

Artemisinin
Artemisinin
Artemisinin
Artemisinin
Artesunate’”

1998 May-Jun
1997
Ott-Dee
1998 Aug-Ott
1998 Ott-Dee

Artemisinin
Artesunate
Artesunate”

10 mg/k/day
Do4
D14
4 mg/k/day
Do & 2 mg/k/day
15 mg/kg/day Do4
20 mg/kg/day Do & 10 mg/kg/day D,-,
20 mg/kg/day Do & 10 mg/kg/day DI-4
10 mglklday
DOA
10 mg/k/dv
DO4
10 mg/k/day
Ds6

10 mg/k/day
h6
10 mg/kg/dv
Do4
2.4 mg/kg/day Do & 1.2 mg/kg/day DIA
10 mg/k/day
Do4
20 mg/kg/day Do & 10 mg/kg/day Dim4
20 mg/kg/day Do & 10 mg/kg/day D,,
2.4 mg/kg/day Do & 1.2 mg/kg/day D,-,

Binh Phuoc Province,
n

%R

102
18
86
50
32
22
15
22
22
30
52

15
12

12
28
34
9
*26
9
9
*26

14
65
28

*E
48
40
14

The possibility that some of these were re-infections cannot be ruled out. Im, intramuscular; iv, intravenous; (C), made in China; *, 21day rather than 28-day follow-up. (N. D. Sy, D. H. Nham, N. M. Huong, N. V. Huong, T. T. Thin, N. V. Thanh, D. X. Huong & T. N.
Hai, unpublished results).


l’LASMODIUM

FALCPARUM

DRUG RESISTANCE IN VIET NAM

V. Thanh reported 100% sensitivity and an IC50 of
340 nmol/L in a study of 3 1 isolates exposed to quinine

and, in 1999, T. T. Tinh reported 100% sensitivity and
an I&, of 142 nmol/L in 19 isolates.
A number of researchers have observed a positive
correlation between responses in vitro to artemisinin (or
its derivatives) and mefloauine (DOURY et al.. 1992:
BASCO & LE BRAS, 1993; -BUST& et al., 1994; RING:
WALD et al.,

1999;

WONGSRICHANALAI

et al., 1999).

Where this pattern is apparent at low ICsos it might be
attributed to the general biological fitness of the isolates,
but when the pattern holds at higher concentrations,
cross-resistance seems more likely. This explanation may
have been applicable to the isolate in our study for which
schizont inhibition in vitro required high concentrations
of both mefloquine and artemisinin. Although, as discussed above, such combination therapy appears mutually protective, there is also a need to sustain regular
in-vitro testing where such a regimen is used so that true
cross-resistance can be identified at an early stage.
Our results have implications for antimalarial prophylaxis and treatment strategies for residents and travellers
to Viet Nam. In addition, they may contribute to our
understanding of the epidemiology of parasite drug
resistance. We have confirmed that artesunate clears
malaria parasites quickly and that it is well tolerated.
Unfortunately the use of artesunate alone is not viable
because of the high recrudescence rates seen with short

treatment regimens. Mefloquine is a drug with a significant side-effect profile and it is also relatively expensive.
Our findings suggest that it may be possible to take
advantage of increased sensitivity of l? falciparum
to
chloroquine by using this drug in combination with
artesunate as an alternative to mefloquine. Although
antagonism in vitro has been reported for chloroquine
plus artemisinin (STAHEL et al., 1988), the effects were
weak and therefore not likely to present a clinical
problem. Unless the antagonism was extreme and resulted in a significant drop in parasite clearance time, the
combination would be likely to be beneficial (WHITE,
1998). Chloroquine-based prophylactic regimens might
also currently afford protection in some endemic areas of
Viet Nam for groups such as migrant workers, pregnant
women and travellers. Nevertheless, further studies are
needed to determine the effectiveness of chloroquine in
viva as part of combination therapy where parasite
sensitivity appears to have been re-established.
Acknowledgements
We thank MS Cath Barker, Dr Le Thi Nga and Dr Phillip
Passmore of VAMCP and Dr Allan Schapira of WHO for their
valuable assistance. This study received financial support from
AusAID (the Australian Agency for International
Developmerit)
References
Barradell, L. B. & Fitton, A. (1995). Artesunate. A review of its
pharmacology and therapeutic efftcacy in the treatment of
malaria. Drugs, 50, 714-741.
Basco, L. K. & Le Bras, J. (1993). In vitro activity of artemisinin
derivatives against African isolates and clones of Plasmodium

falciparum. AmericanJournal of Tropical Medicine and Hygiene,
49,301-307.
Bunnag, D., Viravan, C., Looareesuwan, C., Karbwang, J. &
Harinasuta, T. (199 1). Double blind randomized clinical trial

329

of two different regimens of oral artesunate in falciparum
malaria. SoutheastAsianJoumalof
TropicalMedicine andPublic
Health, 22,534-538.
Bustos, M. D., Gay, F. & Diquet, B. (1994). In-vitro tests on
Philippine isolates of Plasmodium faZcipamm against four
standard antimalarials and four qinghaosu derivatives. Bulletin of the World Health Organization, 72, 729-735.
Doury, J. C., Ringwald, I’., Guelain, J. & Le Bras, J. (1992).
Susceptibility of African isolates of Plasmodium falciparum to
artemisinin (qinghaosu). Tropical Medicine and Parasitology,
43,197-198.
Fevre, E. M., Bamish, G., Yamokgul, I?. & Rooney, W. (1999).
Sensitivitv in vitro of Plasmodium falcinarum to three currentlv
used antimalarial drugs on the western border of Thailand.
Transactions of the Royal Society of Tropical Medicine and
Hygiene, 93,180-184.Hien, T. T., Day, N. I’., Nguyen, H. I’., Nguyen, T. H., Pham,
P. L., Dinh, X. S., Ly, V. C., Ha, V., Waller, D., Peto, T. E. &
White, N. J. (1996). A controlled trial of artemether or
quinine in Vietnamese adults with severe falciparum malaria.
New EnglandJournal of Medicine, 335, 76-83.
Kain, K. C. St Lanar, D. E. (1991). Determination of genetic
variation within Plasmodium falcipamm by using enzymaticallv amnlified DNA from filter uauer disks imnreanated with
whole blood. Journal of ClinicalMhrobiology,

25, 117 1- 1174.
Looareesuwan, S., Viravan, C., Vanijanonta, S., Wilairatana,
I’., Suntharasnmai, P., Charoenlarp, P., Arnold, K., Kyle, D.,
Canfield, C. & Webster, K. (1992). Randomized trial of
artesunate and mefloquine alone and in sequence for acute
uncomplicated falciparum malaria. Lancet, 339, 820-824.
Luxemburger, C., Brockman, A., Silamut, K., Nosten, F., Vugt,
M., Gimenez, F., Chongsuphjaisiddhi,
T. & White, N. J.
(1998). Two patients with falcipamm malaria and poor
responses in vivo to artesunate. Transactions of the Royal
Society of Tropical Medicine and Hygiene, 92, 668-669.
Nguyen, T. A. (1993). Malaria in Vietnam. Environment,
prevention and treatment. Bulletin de la Societe Pathologique
et Exotique, 86, 494-499.
Nosten, F., Hien, T. T. & White, N. J. (1998). Use of
artemisinin derivatives for the control of malaria. Medecine
Troaicale. 58. 45-49.
Phan; V. y. (i998). Epidemiologic du Paludisme et Lutte antipaludique au Vietnam. Hanoi: Editions Medicales Vietnam,
pp. 36.-41.
Ringwald, P., Bickii, J. & Basko, L. K. (1999). In vitro activity of
dihydroartemisinin
against clinical isolates of Plasmodium
falciparum in Yaounde, Cameroon, American Journal of
Tropical Medicine and Hygiene, 61, 187- 192.
Stahel, E., Druilhe, P. & Gentilini, M. (1988). Antagonism of
chloroquine with-other antimalarials. Transactions oythe Royal
Society of Tropical Medicine and Hygiene, 82, 22 1.
Wemsdorfer,
W. H. & Wemsdorfer,

M. G. (1995). The
evaluation of in vitro tests for the assessment of drug. resnonse
in Plasmodium falciparum. Mitteilungen der GsteGeich’ischen
Gesellschafifiir Tropenmedizin und Parasitologic, 17, 22 l-228.
White, N. J. (1998). Preventing antimalarial drug resistance
through combinations. Drug Resistance Updates, 1, 3-9.
WHO (1973). Chemotherapy of Malaria and Resistance to Antimalarials. Repon of a WHO Scientific Working Group. Geneva,
Switzerland: World Health Organization, Technical Report
Series, no. 529.
Wongsrichanalai, C., Wimonwattrawatee,
T., Sookto, P., Laoboonchai, A., Heppner, D. G., Kyle, D. E. & Wernsdorfer,
W. H. (1999). In vitro sensitivity of Plasmodium falciparum to
artesunate in Thailand. Bulletin of the World Health Organization, 77, 392-398.

Received 27 September 2000; revised 12 December 2000;
accepted for publication 13 December 2000