Resistance of plasmodium falciparum to a

Resistance of Plasmodium falciparum to antimalarial drugs in a highly endemic area of southern Viet Nam: a study in viva and in vitro Nguyen Mai Huong’, Sean Hewitt*, Timothy M.. Davis2

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

Nguyen Mai Huong’, Sean Hewitt*, Timothy M E Davis2’3, Le Due Dao’, Tran Quoc Toan’, Tran Bach Kim’, Nguyen Thi Hanh’, Vo Nhu Phuong’ , Doan Hanh Nhan’ and Le Dinh Gong’ ‘Nutianal

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

Keywords: malaria, Plasmodium falciparum, drug sensitivity, in viva, in vitro, chloroquine, mefloquine, quinine, artesunate, artemisinin, Viet Nam

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 evi-

dence 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 En-

tomology (NIMPE) in Hanoi established routine mon-

itoring 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, parti-

cularlv 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 artemisi-

nin 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, Uni-

versity ofwestern Australia, Department of Medicine, Freman-

tle Hospital, P.O Box 480, Fremantle, Western Australia 6959,

Australia; phone +618 9431 3229, fax +618 9431 2977, e-mail

tdavis@cyllene.uwa.edu.au

the light of these reports, and given the history of drug use and parasite resistance in Viet Nam, we aimed to estab- lish 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 28- day 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) concomi- tant illness, and (iii) prior antimalarial therapy, specifi- cally 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 pre-

vious 28 days Dill-Glazko urine tests (reagents pre-

Dared bv NIMPE) were nerformed to confirm that all

recruits had taken neither chloroquine nor sulfadoxine-

pyrimethamine

(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

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 magnifica-

tion) 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 tempera-

ture) 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 follow-

up 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 modifica-

tion of the method described bv KAIN & LANAR C 199 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 protei-

nase 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

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 meflo- quine, chloroquine, and quinine, parasites were consid- ered sensitive if complete inhibition of schizonts occurred at 3200,800 and 2560 nmol/L or less, respec- tively 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 com- parisons were by Student’s t-test or, in the case of non- normally distributed or discontinuous data, by Wilcox- on-Mann-Whitney tests The x2 test was used to assess differences in proportions between groups Associations between variables were assessed using Pearson’s pro- duct-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 dis- tribution, 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%) Symp- toms 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

Sex (% males)

Parasitaemia (/@J 7400 (1040-114000) 18 200 (1560-75 300)*

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

PLASMODIUM FALCIPARUM DRUG RESISTANCE IN VIET NAM 327

120000

.m

E

s 60000 r m"

z 40000

20000

0

16 18 20 22 24 26 28

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)

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

Concentration (nmol/L)

Artemisinin 31 15.0 (9.4-24.1) 84.0 (43.4-162.5) 341 (130-892) Values are geometric means (95% confidence intervals in parentheses)

Among the 32 isolates tested successfully against chlor-

oquine in vitro, 5 (16%) were resistant (discriminating

concentration 80 nmol/L) All 31 isolates tested against

quinine and artemisinin were sensitive to quinine (dis-

criminating concentration 2560 nmol/L) and showed

complete schizont inhibition at artemisinin concentra-

tions >300 nmol/L Further analysis of the in-vitro

results for individual isolates revealed significant correla-

tions 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 me-

floquine group (8%) and in 9 patients from the artesu-

nate group (36%) between 14 and 28 days (P = 0.03)

Cytogenetic studies indicated that both the mefloquine-

treated 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, re-

infected 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 re-

treated with the alternative drug regimen and prompt

parasite clearance was observed in each case

Of the 5 day-0 isolates taken from patients subse-

quently exhibiting recrudescence after artesunate ther-

apy, 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 meflo- quine with complete inhibition of schizont formation occurring only in the well containing 320 nmol/L In 2 patients who did not recrudesce after artesunate, inhibi- tion 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 anti- malarial 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 meflo- quine 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 Antima-

larial 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 (NOS-

TEN 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) Further-

more, 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, unpub-

lished 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

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 combi- nation 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, unpub- lished report) However, the short shelf-life for artemi- sinin 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, there- fore, 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

Table 3 A summary of unpublished studies carried out in Phu Rieng Commune, Binh Phuoc Province, showing fluctuations in recrudescence rates (%R)

199 1 May- Jun Artemisinin

Sep-Nov

15 mg/kg/day Do4

mg/k/day D14

1996 Ott-Dee Artesunate’” 2.4 mg/kg/day Do &

1997

*E

Artesunate”

20 mg/kg/day Do &

10 mg/kg/day Dim4 2.4 mg/kg/day Do &

The possibility that some of these were re-infections cannot be ruled out Im, intramuscular; iv, intravenous; (C), made in China; *, 21- day 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 329

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 dis-

cussed above, such combination therapy appears mu-

tually 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 prophy-

laxis 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 signifi-

cant 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 re-

sulted 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 Develop-

merit)

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