P
l a s m o d iu m f a l c ip a r u m r e s is t a n t t o c h l o r o q u in eAND TO PYRIMETHAMINE IN COMOROS
RANDRIANARIVELOJOSIA M .*,**, RAHERINJAFY R.H.*, MIGLIANI R .*,***, MERCEREAU-PUIJALON O.****, ARIEY F.* & BEDJA S.A.*****
Summary :
W e report the outcom e o f chlo roqu in e treatm ent an d the prevalence o f mutations a t c o d o n 8 6 o f the p fm d r 1 gene, at co d o n 7 6 o f the p fcrt gene, a n d a t co d o n 1 0 8 o f the pfd h fr g e ne in c lin ica l isolates o f Plasm odium fa lcipa rum collected from 3 0 children under 1 0 years o f a g e living in the C om oros U nion.
This in vivo study w a s carrie d out in February a n d M a rc h 2 0 0 1 in M o ro n i. C h lo ro q u in e treatm ent fa ile d in 2 3 children ( 7 6 .6 %;
9 5 % co n fid e n ce interval: 5 7 . 7 to 9 0 .1 %). Subsequent g e n o typ in g show e d that all P. fa lcipa rum isolates ( 1 0 0 %) harboured a tyrosine residue a t position 8 6 in p fM D R l. 8 3 .3 % ( 2 5 / 3 0 ) o f these isolates harboured a mutation a t position 7 6 in pfCRT a n d half ( 1 5 / 3 0 ) o f these isolates also harboured a mutation a t position 1 0 8 in pfDHFR. C h lo ro q u in e resistance is a real concern in the C om oros U nion. The prevalence o f pfDHFR mutant parasites is alarm ing . The alternative drugs p ro po se d as a replacem ent for chlo roqu in e as first-line treatm ent in C om oros, and the strategy to m onitor the dru g susceptibility o f Plasm odium sp in this pa rt o f the Indian O c e a n sub-region a re discussed.
KEY WORDS : Comoros, chloroquine, malaria, Plasmodium falciparum, resistance.
R ésu m é : Ré s is t a n c e d e Pla sm o d iu mfa lc ip a r u mà la c h l o r o q u in e ET À LA PYRIMETHAMINE AUX COMORES
C e t article d é c rit la réponse thérapeutique à la chlo roqu in e dans le traitem ent du pa lu dism e à P. fa lcipa rum ch e z 3 0 enfants d e moins d e 10 ans atteints d e paludism e, vivant dans l'U nio n des Com ores, et aussi la pré va len ce des mutations au niveau des marqueurs génétiques d e la résistance des parasites à la chlo roqu in e (pfcrt K76T, pfm dr 1 N 8 6 Y ), et d e la résistance à la pyrim étham ine (pfdhfr S 1 0 8 N ) ch e z les isolats d e P. falciparum prélevés à J0. Lors d e l'é lu d e réalisée en février e t mars 2 0 0 1 à M o ro n i, le traitem ent p a r chlo roqu in e en m onothérapie a échoué ch e z 2 3 enfants (7 6 , 6 %; Intervalle d e c o n fia n ce 9 5 % : 5 7 ,7 - 9 0 , 1 %), La détection des mutations p a r la technique d e PCR/RFLP a montré que tous les isolats d e P. falciparum lestés ( 1 0 0 %) contiennent la tyrosine à la po sitio n 8 6 dans pfM D R 1 . 8 3 ,3 % (2 5 / 3 0 ) d e ces isolais contenaient des souches mutées à la po sitio n 7 6 p o u r pfC R T e l la m oitié ( 1 5 / 3 0 ) des isolats était mutée a s p a ra g in e -10 8 pfdhfr. Les résultats des tests in vivo et le g é n o ty p a g e m ontrent que la résistance d e P. falciparum à la chlo roqu in e est à présent un p ro b lè m e m ajeur dans l'U nio n des C om ores. La pré va len ce des parasites mutés p o u r pfDHFR, d o n c potentiellem ent résistants à la pyrim étham ine, est alarm ante. Les propositions des alternatives à la chlo roqu in e p o u r le traitem ent de prem ière intention dans l'U nio n des C om ores, et la stratégie de surveillance d e la sensib ilité/ré sista nce d e Plasmodium sp. dans celle sous-région d e l ’O c é a n Indien sont discutées.
MOTS CLÉS : Comores, chloroquine, paludisme, Plasmodium falciparum, résistance.
I
n Comoros Union, 15 to 30 % of hospital admissions are due to malaria, and malaria is responsible for 15 to 20 % of all deaths registered in paedia
tric units (Ouledi, 1995). P la sm o d iu m fa lc ip a r u m accounts for over 95 % of malaria cases. Chloroquine
* Groupe de Recherche sur le Paludisme, Institut Pasteur de Madagas
car, BP 1274 Antananarivo (101), Madagascar.
** Natural Products Research Group, School o f Chemistry, Univer
sity o f KwaZulu-Natal, Durban, 4041, South Africa.
*** Service de Médecine des collectivités, Institut de Médecine Tro
picale du Service de Santé des Armées, Marseille, France.
**** Unité d'immunologie Moléculaire des Parasites, Institut Pasteur, 25 rue du Dr Roux, 75015, Paris, France.
***** Direction de Lutte contre les Endémies et les Epidémies, BP 2068, Moroni, Union des Comores.
Correspondence : Dr Milijaona Randrianarivelojosia, Groupe de Recherche sur le Paludisme, Institut Pasteur de Madagascar, BP 1274, Antananarivo (101), Madagascar.
Fax: +261 20 22 415 34 - E-mail: milijaon@pasteur.mg
has been the recommended first-line treatment for mala
ria for the last four decades. Local populations with limited income, frequently use a chloroquine auto-medi
cation in case of fever. Recent reports from peripheral health districts of the archipelago (Grande Comore, Anjouan and Mohéli) have shown an increasing rate of chloroquine treatment failure, which now exceeds 30 % (Silai, unpublished data). This probably reflects increasing prevalence of chloroquine-resistant P. f a l ciparu m parasites in the Comoros. However, in order to change the national treatment policy, evidence- based information is needed.
Within the frame of a regional network on malaria, the Institut Pasteur de Madagascar participated in an in vivo evaluation of chloroquine effectiveness for the treat
ment of uncomplicated fa lc ip a ru m malaria in Moroni (Grande Comore) in 2001. This study was the first step of a regional collaboration between Madagascar and
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2004114419
RANDR1 ANARTVELOJOSLA M„ RAHERINJAFY R.H., MIGLIANI R. E T A L.
Comoros. Above all, the aim was to document chlo- roquine effectiveness and the prevalence of the pfCRT mutation K76T associated in other settings with chlo- roquine-resistance (Fidock et al., 2000; Djimde et al., 2001).
MATERIALS AND METHODS
In vivo t e s t a n d c o l l e c t i o n o f b l o o d sa m p le
T
he in vivo efficacy of chloroquine treatment in children with uncomplicated fa lcip a ru m malaria was measured in Moroni in March 2001. The study was approved by the Ethics Committee of the Comorian Ministry of Health. Informed consent was obtained from all parents or guardians. Thirty-two children with uncomplicated P. fa lc ip a r u m malaria were included. Two were excluded during the follow- up, because they were given a quinine injection on Day 1 or Day 2 by a private health worker in their village with parental consent because they had fever (“hot skin” according to local terminology). Thirty children (nine females and 21 males) aged between eight months and nine years (mean age: 3 .6 years; median age: 3.25 years; 95 % confidence interval: 2.78-4.42 years) were monitored for 14 days following WHO protocol (WHO, 1996). Before the administration of chloroquine on Day 0, a fingerprick blood sample was collected on filter paper. The air-dried filter paper was placed in an individual plastic bag and shipped to Institut Pasteur de Madagascar. The samples were kept at - 20° C until use.
DNA EXTRACTION AND PCR (POLYMERASE CHAIN r e a c t i o n )
Mutations in the pfcrt and in pfm dr1 genes were detected by the Malaria Research Group at the Institut Pasteur de Madagascar in October 2002. P. fa lcip aru m DNA was extracted from the filter blood spot. A ~ 10 mm square of blood impregnated filter paper was placed in 200 μl of PBS buffer (pH 7.2) containing 0.5 % Tween 20 (v/v) for three hours at room temperature. The sample was shaken for 10 seconds every hour by use of a vortex mixer. The tube was then centrifuged at 360 g for two minutes at room temperature. The supernatant was col
lected and used for phenol-chloroform DNA extraction as described (Ariey et a l ., 1999). DNA was re-sus- pended in 30 μl of sterile water.
The p fcrt, p fm d r1 and p fd h fr genes were amplified by nested PCR, as described (http://medschool.umary- land.edu/cvd/2002_pcr_asra.html). For each PCR and each digestion, DNA from P. fa lc ip a ru m FCM29 (chlo- roquine-resistant) and 3D7 (chloroquine-sensitive), which are maintained in continuous culture in the laboratory,
was used as a positive control and H2O as a negative control.
Re s t r ic t io n f r a g m e n t l e n g t h p o l y m o r p h is m
The p fcrt and p fm d r1 nested PCR products were diges
ted with A poI, with Afl III (New England Biolabs, UK) respectively, and p fd h fr nested PCR were digested separately with AluI, SrcFI and BsrI (New England Biolabs, UK), in a final volume of 25 μl as described (http://medschool.umaryland.edu/cvd/2002_pcr_asra.htm;
Djimde et al., 2001; Rason et al., 2002; von Seidlein et al., 1997; Zindrou et al., 1996).
The restricted products (15 μl) were subjected to elec
trophoresis in a 2 % agarose gel, stained with 0.5 pg/ml ethidium bromide and visualized under ultraviolet light. The 145-bp pfcrt PCR product contains one A poI site when the codon 76 of the pfcrt gene codes for a lysine (K76), visualised by presence of a 99-bp and a 46-bp restriction fragments. The 291-bp p fm d r 1 PCR product contains a single AflIII site when codon 86 is a mutant-type coding for tyrosine (86Y), resulting in generation of a 165-bp and a 126-bp fragment. The 256-bp p fd h fr PCR products contained one restriction site for either Alul, Bsrl or ScrFI, depending on the amino acid residue at position 108 of the p fd h fr g en e.
AluI, Bsrl and ScrFI each cleaved the PCR product into one 210-bp product and one 46-bp product. AluI cut the product when the wild-type codon was present at position 108 (serine-108), Bsrl cut the product when an asparagine codon was present at position 108 (aspa- ragine-108) linked to pyrimethamine resistance and ScrFI cut the product when a threonine codon was pre
sent at position 108 (threonine-108) linked to cyclo- guanil resistance (Bzik et al., 1987; Cowman et al.,
1988
)RESULTS
Ou t c o m e o f c h l o r o q u in e t r e a t m e n t
O
n Day 0, parasitaemia ranged from 1,560 to 101,000 trophozoites per 8,000 white blood cells (geometric mean parasitaemia: 12,336;95 % confidence interval: 13,800-33,852). There were 21 clinical failures, and two parasitological failures on Day 14 (Table I). Thus, the overall chloroquine treat
ment failure rate was 76.6 % (95 % confidence interval:
57.7-90.1 %). For 15 of 21 children aged below five years (71.4 %), a clinical failure was recorded and in eight cases, treatment failed at an early stage. All chil
dren in whom the initial chloroquine treatment failed were subsequently treated successfully either with qui
nine or with sulfadoxine-pyrimethamine (data not shown).
4 2 0 Parasite, 2004, 11, 419-423
Chloroquineandpyrimethamineresistancein Comoros
C h lo roq u in e tre a tm e n t o u tco m e N um ber o f ch ild ren
C h ildren ca rry in g p arasites w ith m u tatio n at
codon 76 in pfcrt codon 108 in p fd h fr
Overall clinical failure* 21 18 10
Parasitological failure on Day 14 2 2 1
Adequate clinical and parasitological response** 7 5 4
Total 30 (100 %) 25 (83.3 %) 15 (50 %)
Gen o typin g
The 30 nested PCRs for pfcrt, p fm d r l and p fd h fr were successful. All of the examined isolates of P. f a l c i p a ru m collected on day-0 yielded a p fm d r 1 PCR pro
duct that was digested with AflIII. This indicated that all isolates contained the p fm d r 1 tyrosine-86 (86Y) mutation. Only two of 30 isolates were mixed i.e.
contained in addition to the mutant p fm d r l 86Y para
sites, parasites that had an A flI II-insensitive product.
Twenty-five (83-3 %) of the p fc rt PCR products were not cut by ApoI, indicating that they harboured a muta
tion at codon 76. Fifteen (50 %) of the 30 typed iso
lates contained the p fd h fr asparagine-108 mutation, and 11 of these 15 isolates harbouring asparagine-108 p fd h fr harboured also a mutation at pfcrt codon 76.
DISCUSSION
W
e found that the chloroquine treatment failure rate was unacceptably high (76.6 %) in children. The situation seen in Moroni can probably be generalised throughout the archipelago due to the population dynamics between islands.
Almost 20 years ago, a case of chloroquine treatment failure was reported in Germany in a patient who had recently visited Comoros (Eichenlaub & Pohle, 1980).
In the 1980s and early 1990s, the prevalence of chlo
roquine resistance was considered to be low in Como
ros even though RIII resistance cases had been recor
ded (Blanchy & Benthein, 1989; Feillet et a l., 1993).
The selection of chloroquine-resistant P. fa lc ip a r u m is probably related to the high chloroquine consumption by the local inhabitants (Feillet et al., 1993). This pro
bably explains why the treatment failure rate increases in general population (Blanchy & Benthein, 1989;
Ariey et al, 2002).
Even though different studies have concluded that there is not a perfect correlation between the presence of the mutation at position 76 of pfCRT and the cli
nical response to chloroquine treatment (Djimde et al.,
2001; Kyosiimire-Lugemwa et al., 2002; Talisuna et a l., 2002), the prevalence of chloroquine treatment failure is consistently higher when the prevalence of the p fcrt threonine-76 mutation is high. Such situation is now occurring in Comoros. Prior the discovery of pfcrt, the majority of research into the genetic basis of P. fa lc ip a rum chloroquine-resistance focused on p fm d r l (Foote et al., 1990). But it was also reported later that there is no strong correlation between point mutations in p fm d r l gene and clinical response to therapy with chloroquine (Dorsey et al., 2001). Although the role of mutations in known resistance genetic markers in mediating responses after chloroquine therapy remains uncertain, both in vivo test and genotyping reported herein indicate a high prevalence of chloroquine resis
tance in Comoros. Given this dramatic situation, there is an urgent need to reconsider the national malaria control policy and introduce the use of preferment the
rapy based on the use of antimalarial drugs other than chloroquine.
For many years, sulfadoxine-pyrimethamine has been advocated as the second line treatment in Comoros.
This drug has been widely used with or without medi
cal prescription in Moroni (and elsewhere in Comoros).
Our data indicate that a large number of P. fa lc ip a - rum isolates (15/30) already harboured an asparagine- 108 p fd h fr allele that is associated with pyrimethamine resistance (Peterson et al, 1888; Cowman et a l., 1988;
Hyde, 1990; Ranford-Cartwright et al., 2002). We would not give much weight to the prevalence of asparagine- 108, as this single mutation has not been found to be predictive of sulfadoxine-pyrimethamine treatment fai
lure, and the quintuple DHFR/DHPS mutant is the most strongly associated with sulfadoxine-pyrimethamine failure. (Kublin et al., 2002) But for a regional system for malaria resistant surveillance, the occurrence of pre
valent asparagine-108 mutant parasites is alarming. An area with ~ 50 % asparagine-108 is generally likely to have more sulfadoxine-pyrimethamine resistance soo
ner or later. Thus, whatever the profiles (wild or mutant) of the other codons known as pyrimethamine resistance markers in p fd h fr or sulfadoxine resistance
* : Overall clinical failure com bines early and late treatment failure.
** : Clinical and parasitological response means clinical cure and absence o f parasitaemia.
Table I. - Outcome of chloroquine treatment in Comorian children and prevalence o f Plasm odium falcip aru m with mutation at codon 76 in pfcrt (samples were collected on Day 0).
RANDRIANARIVELOJOSIA M., RAHERINJAFY R.H., MIGUANI R. E T A L .
markers in p fd h p s, this presence of prevalent aspara- gine-108 p fd h fr in Comoros is not in favour of a switch from chloroquine to sulfadoxine-pyrimethamine alone (monotherapy) as first line treatment in this archipe
lago. Therefore, it was decided by Comorian health policy makers that combination therapies will be tested to determine an effective replacement for chloroquine.
Fight against malaria should not be limited to a single country. In contrast with the situation in Comoros, P. fa lc ip a r u m isolates carrying p fcrt and p fd h fr muta
tions have been absent (or might be present at very low prevalence) in Madagascar and the clinical res
ponses to chloroquine and to sulfadoxine-pyrimetha- mine are satisfactory (Ariey et al., 2002; Randrianari- velojosia et al., 2002a, 2002b, 2002c; Rason et al., 2002). The dissemination of resistant parasites from an area of resistance to an area of sensitivity is a factor leading to the spread of resistance. Thus, the biggest danger for Madagascar may be importation of resistant malaria in view of the improving commercial exchange between neighbouring islands in this part of the Indian Ocean. However, the Malagasy recording system does not yet allow us to document cases of malaria imported into Madagascar, while for example, it has been proven that many cases of malaria are introduced into France from Comoros (Minodier et al., 1999).
Regional surveys involving the Comoros archipelago and Madagascar should be strengthened to generate up-to-date and comparative in vivo and in vitro data to allow the design of regional and national evidence- based strategies for controlling malaria.
ACKNOWLEDGEMENTS
W
e are indebted to the medical teams from the malaria research group of the Institut Pasteur de Madagascar and from the Ministry of Health in Moroni, Comoros. We thank Drs Marie Ange Rason and Louise Ranaivo, and Mrs Rahamatou Silai for technical assistance. This work was supported by the French Ministry of Co-operation (grant FSP-IG99004900), by the Prix Louis D grants of French Academy of Sciences, and partly by the WHO office in the Comoros Union and the International Atomic Energy Agency as part of the Raf 6025 project.
REFERENCES
Ar i e y F., Ch a l v e t W ., Ho m m e l D ., Pe n e a u C ., Hu l in A ., Me r- c e r e a u- Pu ija l o n O., Du c h e m inJ.B., Sa r t h o uJ.L., Re y n e s J .M .
& Fa n d e u r T. Plasmodium falciparum parasites in French Guiana: limited genetic diversity and high selfing rate.
American Journal o f Tropical Medicine an d Hygiene, 1999, 61, 978-985.
Ar ie y F., Ra n d r ia n a r iv e l o jo s ia M ., Du c h e m in J . B . , Ra k o t o n- d r a m a r in a D ., Ou l e d i A ., Ro b e r t V ., Ja m b o u R ., Ja h e v it r a M ., An d r ia n a n t e n a in a H., Ra h a r im a l a l aL. & Ma u c l e r e P. Plas
modium falciparum pfcrt K76T mutation mapping: a useful tool for malaria control strategy in Madagascar. Journal o f Infectious Diseases, 2002, 185, 710-712.
Bl a n c h y S. & Be n t h e in F. In vivo chemosensitivity of Plas
modium falciparum in the Islamic Federal Republic of the Comoros. Bulletin de la Société de Pathologie Exotique, 1989, 82, 493-502.
Bz ik D.J., Li W . B . , Ho r ii T. & In s e l b u r gJ. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Procee
dings o f the National Academy o f Sciences USA, 1987, 84, 8360-8364.
Co w m a nA.F., Mo r r y M .J ., Bi g g s B.A., Cr o s s G.A. & Fo o t e S.J.
Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proceedings o f the National Academy o f Sciences USA, 1988, 85, 9109-9113.
Djim d e A., Do u m b o O.K., Co r t e s e J . F . , Ka y e n t a oK., Do u m b o S ., Dio u r t e Y., Dic k o A., Su X.Z., No m u r a T., Fi d o c k D .A ., We l l e m s T.E., Pl o w e C.V. & Co u l ib a l y D . A molecular marker for chloroquine-resistant falciparum malaria. New England Journal o f Medicine, 2001, 344, 257-263.
Do r s e y G., Ka m y aM.R., Sin g h A . & Ro s e n t h a l P.J. Polymor
phisms in the Plasmodium falciparum pfcrt and pfmdr-1 genes and clinical response to chloroquine in Kampala, Uganda. Journal o f Infectious Diseases, 2001, 183, 1417- 1420.
Eic h e n l a u b D. & Po h l e H.D. A case of chloroquine-resistant (R1) falciparum malaria from the East African Comoros Islands. Infection, 1980, 8, 90-92.
Fe il l e tN, Ag n a m e y P., Br a s s e u rP. & Dr u il h e P. In vivo resis
tance of P la sm o d iu m fa lc ip a r u m to chloroquine in Anjouan (Comoros). B u lletin d e la S o c ié té d e P a th o lo g ie E x otiq u e, 1993, 86, 48-51.
Fid o c k D.A., No m u r a T . , Ta l l e yA.K., Co o p e r R.A., Dz e k u n o v S .M., Fe r d i g M.T., Ur s o s L.M., Sid h u A.B., Na u d e B.,
De it s c h K.W., S u X.Z., Wo o t t o n J . C . , Ro e p e P.D. & We l
l e m sT.E. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Molecular Cell, 2000, 6, 861-871.
Fo o t e S.J., Ky l e D.E., Ma r t in R .K ., Od u o l a A .M ., Fo r s y t h K ., Ke m p D.J. & Co w m a n A .F . Several alleles of multidrug-resis- tance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature, 1990, 345, 255-258.
Hy d eJ.E. The dihydrofolate reductase-thymidylate synthetase
gene in the drug resistance of malaria parasites. Pharma- cology an d Therapeutics, 1990, 48, 45-59.
Ky o s ii m i r e-Lu g e m w a J., Na l u n k u m a- Ka z ib w e A.J., Mujuzi G ., Mu l in d w a H., Ta l isu n a A. & Eg w a n g T . G . The Lys-76-Thr mutation in P f C R T and chloroquine resistance in Plasmo
dium falciparum isolates from Uganda. Transactions o f the Royal Society o f Tropical Medicine an d Hygiene, 2002, 96, 91-95.
Ku b l inJ.G., Dz in ja l a m a l a F .K ., Ka m w e n d o D . D . , Ma l k in E.M.,
Co r t e s e J . F . , Ma r t in o L .M ., Mu k a d a m R .A ., Ro g e r s o n S.J.,
4 2 2 Parasite, 2004, 11, 419-423
Ch lo r o q u in e an d pyr im e th a m in e resista n ce in Co m o r o s
Le s c a n o A.G., Mo l y n e u x M.E., Win s t a n l e yP.A., Ch im p e n iP.,
Ta y l o rT.E. & Pl o w e C.V. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. Journal o f Infectious Diseases, 2002, 185, 380-388.
Min o d ie r P . , La n z a- Sil h o l F ., Pi a r r o u x R ., Ga r n ie r J . M . , Du m o n H. & Un a l D. Imported pediatric malaria in Mar
seille. Archives de Pediatrie , 1999, 6, 935-943.
Ou l e d i A . Epidemiology and control of malaria in the Federal Islamic Republic of Comoros. Cahiers Santé, 1995, 5, 368- 371.
Pe t e r s o n D.S., Wa l l ik e r D. & We l l e m s T.E. Evidence that a point mutation in dihydrofolate reductase-thymidylate syn
thase confers resistance to pyrimethamine in falciparum malaria. Proceedings o f the National Academy o f Sciences USA, 1988, 85, 9114-9118.
Ra n d r ia n a r iv e l o jo s ia M ., Sa h o n d r a-Ha r is o a J .L . , Ra b a r ija o n a L .P ., Ra h a r im a l a l a L .A ., Ra n a iv o L .H ., Pie t r a V ., Du c h e m in J . B . , Ra k o t o m a n a n a F., Ro b e r t V ., Ma u c l e r e P . & Ar ie y F.
In vitro sensitivity of Plasmodium falciparum to amodia- quine compared with other major antimalarials in Mada
gascar Parassitologia, 2002. 44, 141-147.
Ra n d r ia n a r iv e l o jo s iaM., Ar ie y F ., Ra h a r im a la laL.A., Pa r z yD.,
Ro g i e r C. & Ja m b o u R . Current absence of pyrimethamine- resistance of Plasmodium falciparum in Madagascar. Tran
sactions o f the Royal Society o f Tropical Medicine an d Hygiene, 2002b, 96, 557-559.
Ra n d r ia n a r iv e l o jo s ia M., Ra t s im b a s o a A., Ra n d r ia n a s o l o L ., Ra n d r ia n a r ija o n aA. & Ja m b o u R . In vitro sensitivity of Plas
modium falciparum to chloroquine, halofantrine, meflo
quine and quinine in Madagascar. East African Medical Journal. 2002c, 79, 237-241.
Ra n f o r d- Ca r t w r ig h tL.C., Jo h n s t o n K .L ., Ab d e l- Mu h s inA.M.,
Kh a n B .K . & Ba b ik e r H .A . Critical comparison of molecular genotyping methods for detection of drug-resistant Plas
modium falciparum. Transactions o f the Royal Society o f Tropical Medicine an d Hygiene, 2002, 96, 568-572.
Ra s o n M.A., Ar i e y F., Ra f id im a n a n t s o a L., An d r ia n a n t e n a in a
B.H., Sa h o n d r a- Ha r is o a J .L . & Ra n d r ia n a r i v e l o jo s ia M.
Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro che- mosensitivity and mutation detection tests. Parasite, 2002, 9, 247-253.
Ta l is u n a A .O .. Ky o s ii m i r e-Lu g e m w a J . , La n g i P .. Mu t a b in g w a T .K ., Wa t k in s W . , Va n Ma r c k E ., Eg w a n g T . & D 'Al e s- s a n d r o U. Role of the pfcrt codon 76 mutation as a mole
cular marker for population-based surveillance of chloro
quine (CQ)-resistant Plasmodium falciparum malaria in Ugandan sentinel sites with high CQ resistance. Transac
tions o f the Royal Society o f Tropical Medicine and Hygiene, 2002, 96, 551-556.
v o n Se id l e in L, Du r a is in g h M.T., Dr a k e l e y C .J ., Ba il e y R.,
Gr e e n w o o d B.M. & Pin d e r M. Polymorphism of the Pfmdr1 gene and chloroquine resistance in Plasmodium falci- parum in The Gambia. Transactions o f the Royal Society
o f Tropical Medicine an d Hygiene, 1997, 91, 450-453.
Zin d r o u S ., Da o L .D ., Xu y e n P.T., Du n g N.P., Sy N .D ., Sk o l dO.
& Sw e d b e r g G. Rapid detection of pyrimethamine suscep
tibility of Plasmodium falciparum by restriction endonu-
clease digestion of the dihydrofolate reductase gene. Ame
rican Journal o f Tropical Medicine an d Hygiene, 1996, 54, 185-188.
WHO. Assessment o f therapeutic efficacy o f antim alarial drugs fo r uncomplicated falciparum malaria in area with intense transmission. Geneva: World Health Organization.
WHO/MAL96.1077, 1996.
Reçu le 4 février 2004 Accepté le 2 avril 2004
