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    A plot of the IC50 for primary and recrudescent isolates for artemisinin (A), mefloquine (B), chloroquine (C) and quinine (D). Each dot corresponds to the IC50 of a particular isolate and the line signifies the mean of the isolates. The arrow in B, C and D refers to the threshold of resistance obtained previously by determining serum drug concentration in patients infected with sensitive and resistant forms of P. falciparum.

  • 1

    WHO, 1997. Malaria in the South-East Asia Region, Regional office for South-East Asia, New Delhi.

  • 2

    Phan VT, 1990. Technical problems and solution of malaria eradication in Vietnam. Vietnamese scientific research report period 1986–1990 (NIMPE, Vietnam). 9–21.

  • 3

    Cong LD, Sy ND, Hinh TD, Huong NV, Thanh NV, Tien NT, Van Manh DH, 1994. Study on appropriate solutions for control of malaria parasite drug - resistance and vector prevention. Information for the control and prevention of malaria in Vietnam. 2 :1–5.

    • Search Google Scholar
    • Export Citation
  • 4

    Zindrou S, Dung NP, Sy ND, Skold O, Swedberg G, 1996. Plasmodium falciparum: mutation pattern in the dihydrofolate reductase-thymidylate synthase genes of Vietnamese isolates, a novel mutation, and coexistence of two clones in a Thai patient. Exp Parasitol 84 :56–64.

    • Search Google Scholar
    • Export Citation
  • 5

    Am NT, 1993. Malaria in Vietnam—environment, prevention and treatment. Bull Soc Pathol Exot 86 :494–499.

  • 6

    Krogstad DJ, Gluzman IY, Kyle DE, Oduola AM, Martin SK, 1987. Efflux of chloroquine from Plasmodium falciparum: mechanism of chloroquine resistance. Science 238 :1283–1285.

    • Search Google Scholar
    • Export Citation
  • 7

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

    • Search Google Scholar
    • Export Citation
  • 8

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

    • Search Google Scholar
    • Export Citation
  • 9

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

    • Search Google Scholar
    • Export Citation
  • 10

    Harinasuta T, Viravan C, Reid HA, 1967. Sulphormethoxine in chloroquine-resistant falciparum malaria in Thailand. Lancet 1 :1117–1119.

  • 11

    Harinasuta T, Viravan C, Buranasin P, 1988. Parenteral Fansidar in falciparum malaria. Trans R Soc Trop Med Hyg 82 :694.

  • 12

    Cowman AF, Morry MJ, Biggs BA, Cross GAM, Foote SJ, 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci U S A 85 :9109–9113.

    • Search Google Scholar
    • Export Citation
  • 13

    Peterson DS, Walliker D, Wellems TE, 1988. Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc Natl Acad Sci U S A 85 :9114–9118.

    • Search Google Scholar
    • Export Citation
  • 14

    Sirawaraporn W, Yuthavong Y, 1984. Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi. Mol Biochem Parasitol 10 :355–367.

    • Search Google Scholar
    • Export Citation
  • 15

    Wu Y, Kirkman LA, Wellems TE, 1996. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc Natl Acad Sci U S A 93 :1130–1134.

    • Search Google Scholar
    • Export Citation
  • 16

    Basco LK, de Pecoulas PE, Wilson CM, Le Bras J, Mazabraud A, 1995. Point mutations in the dihydrofolate reductase-thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum. Mol Biochem Parasitol 69 :135–138.

    • Search Google Scholar
    • Export Citation
  • 17

    Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, Winstanley PA, Estrada-Franco JG, Mollinedo RE, Avila JC, Cespedes JL, Carter D, Doumbo OK, 1997. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance. J Infect Dis 176 :1590–1596.

    • Search Google Scholar
    • Export Citation
  • 18

    Nagesha HS, Din-Syafruddin D, Casey GJ, Susanti AI, Fryauff DJ, Reeder JC, Cowman AF, 2001. Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in vivo drug resistance in West Papua, Indonesia. Trans R Soc Trop Med Hyg 95 :1–7.

    • Search Google Scholar
    • Export Citation
  • 19

    Triglia T, Cowman AF, 1994. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc Natl Acad Sci U S A 91 :7149–7153.

    • Search Google Scholar
    • Export Citation
  • 20

    Brooks D, Wang P, Read M, Watkin W, Sims P, Hyde J, 1994. Sequence variation in the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine. Eur J Biochem 224 :397–405.

    • Search Google Scholar
    • Export Citation
  • 21

    Triglia T, Menting JGT, Wilson C, Cowman AF, 1997. Mutations of dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A 94 :13944–13949.

    • Search Google Scholar
    • Export Citation
  • 22

    Triglia T, Wang P, Sims PFG, Hyde JE, Cowman AF, 1998. Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria. EMBO J 17 :3807–3815.

    • Search Google Scholar
    • Export Citation
  • 23

    Wang P, Read M, Sims PF, Hyde JE, 1997. Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization. Mol Microbiol 23 :979–986.

    • Search Google Scholar
    • Export Citation
  • 24

    Hien TT, VinhChau NV, Vinh NT, Hunh NT, Phung MY, Toan LM, Mai PP, Dung NT, HoaiTam DT, Arnold K, 1997. Management of multiple drug-resistant malaria in Vietnam. Ann Acad Med 26 :659–663.

    • Search Google Scholar
    • Export Citation
  • 25

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193 :673–675.

  • 26

    Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16 :710–718.

    • Search Google Scholar
    • Export Citation
  • 27

    Duraisingh MT, Curtis J, Warhurst DC, 1998. Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp Parasitol 89 :1–8.

    • Search Google Scholar
    • Export Citation
  • 28

    Duraisingh MT, Roper C, Walliker D, Warhurst DC, 2000. Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum. Mol Microbiol 36 :955–961.

    • Search Google Scholar
    • Export Citation
  • 29

    Felger I, Tavul L, Beck H-P, 1993. Plasmodium falciparum: a rapid technique for genotyping the merozoite surface protein 2. Exp Parasitol 77 :372–375.

    • Search Google Scholar
    • Export Citation
  • 30

    Duraisingh MT, Drakeley CJ, Muller O, Bailey R, Snounou G, Targett GA, Greenwood BM, Warhurst DC, 1997. Evidence for selection for the tyrosine-86 allele of the pfmdr 1 gene of Plasmodium falciparum by chloroquine and amodiaquine. Parasitology 114 :205–211.

    • Search Google Scholar
    • Export Citation
  • 31

    Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE, 1995. Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52 :565–568.

    • Search Google Scholar
    • Export Citation
  • 32

    Plowe CV, Djimde A, Wellems TE, Diop S, Kouriba B, Doumbo OK, 1996. Community pyrimethamine-sulfadoxine use and prevalence of resistant Plasmodium falciparum genotypes in Mali: a model for deterring resistance. Am J Trop Med Hyg 55 :467–471.

    • Search Google Scholar
    • Export Citation
  • 33

    Plowe CV, Kublin JG, 1998. P. falciparum DHFR and DHPS mutations: epidemiology and role in clinical resistance to antifolates. Drug Resistance Updates.

  • 34

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

    • Search Google Scholar
    • Export Citation
  • 35

    Foote SJ, Thompson JK, Cowman AF, Kemp DJ, 1989. Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum. Cell 57 :921–930.

    • Search Google Scholar
    • Export Citation
  • 36

    Sirawaraporn W, Sirawaraporn R, Cowman AF, Yuthavong Y, Santi DV, 1990. Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum. Biochemistry 29 :10779–10785.

    • Search Google Scholar
    • Export Citation
  • 37

    Wang P, Read M, Sims PFG, Hyde JE, 1997. Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilisation. Mol Microbiol 23 :979–986.

    • Search Google Scholar
    • Export Citation
  • 38

    Morillon M, Baudon D, Dai B, 1996. Malaria in Vietnam in 1996: brief synthesis of epidemiological data. Med Trop 56 :197–200.

  • 39

    Basco LK, Le Bras J, 1993. In vitro activity of artemisinin derivatives against African isolates and clones of Plasmodium falciparum. Am J Trop Med Hyg 49 :301–307.

    • Search Google Scholar
    • Export Citation
  • 40

    Pradines B, Rogier C, Fusai T, Tall A, Trape JF, Doury JC, 1998. In vitro activity of artemether against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial drugs. Am J Trop Med Hyg 58 :354–357.

    • Search Google Scholar
    • Export Citation
  • 41

    Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 1999. Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study. Parasitology 119 :435–440.

    • Search Google Scholar
    • Export Citation
  • 42

    Barnes DA, Foote SJ, Galatis D, Kemp DJ, Cowman AF, 1992. Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum. EMBO J 11 :3067–3075.

    • Search Google Scholar
    • Export Citation
  • 43

    Peel SA, Bright P, Yount B, Handy J, Baric RS, 1994. A strong association between mefloquine and halofantrine resistance and amplification, overexpression, and mutation in the Pglycoprotein gene homolog (pfmdr) of Plasmodium falciparum in vitro. Am J Trop Med Hyg 51 :648–658.

    • Search Google Scholar
    • Export Citation
  • 44

    Wang P, Sims PFG, Hyde JE, 1997. A modified in vitro sulfadoxine susceptibility assay for Plasmodium falciparum suitable for investigating Fansidar resistance. Parasitology 115 :223–230.

    • Search Google Scholar
    • Export Citation

 

 

 

 

ANALYSIS OF PFCRT, PFMDR1, DHFR, AND DHPS MUTATIONS AND DRUG SENSITIVITIES IN PLASMODIUM FALCIPARUM ISOLATES FROM PATIENTS IN VIETNAM BEFORE AND AFTER TREATMENT WITH ARTEMISININ

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  • 1 Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; University of Melbourne, Parkville; MacFarlane Burnet Centre for Medical Research, Melbourne, Australia; National Institute of Malariology, Parasitology, and Entomology, Hanoi, Vietnam

We have analyzed artemisinin sensitivity in Plasmodium falciparum isolates obtained from patients in South Vietnam and show that artemisinin sensitivity does not differ before and after drug treatment. There was an increase in the level of mefloquine resistance in the isolates after drug treatment that was concomitant with a decrease in chloroquine resistance, suggesting that treatment with artemisinin has selected for increased mefloquine resistance. Mutations in the pfmdr1 gene, previously shown to be associated with sensitivity to mefloquine, were selected against. All isolates resistant to chloroquine encoded Thr-76 in the pfcrt gene consistent with an essential role in the mechanism of chloroquine resistance. Mutations in pfmdr1 also were linked to chloroquine resistance. High levels of mutation in dhfr and dhps genes, which have previously been associated with Fansidar resistance, also were found, suggesting that this drug would not be useful for malaria control in this part of Vietnam.

INTRODUCTION

The development and spread of drug resistance in Plasmodium falciparum is a major problem for the treatment and control of malaria in many endemic countries. In Southeast Asia, 21.9 million cases of malaria were reported in 1995 alone.1 In Vietnam, chloroquine resistance was first reported in 1961 and reached a peak in 1990, ranging in incidence from 60–80% in some regions.2 Chloroquine resistance has now spread to most regions of Vietnam where malaria is endemic3 and has led to the widespread use of alternative antimalarial drugs (mefloquine, halofantrine, artemisinin derivatives, and pyrimethamine/sulfadoxine). Use of Fansidar (pyrimethamine + sulfadoxine) has increased rapidly over the last 30 years.4 As a consequence, reported levels of resistance to the drug have increased from approximately 20% to over 80%.5 Since 1991, artemisinin and its derivatives have been used to treat malarial patients in Vietnam. These drugs have become increasingly important as antimalarial therapy in this setting because of their rapid mode of action and efficacy against multidrug-resistant forms of P. falciparum malaria.

Resistance of P. falciparum to chloroquine has been associated with lower drug accumulation.6 Mutations in the pfcrt gene have been strongly linked to the mechanism of chloroquine resistance.7 The presence of Tyr-76 residue within the Pfcrt protein is linked to chloroquine resistance, suggesting that it plays an important role in the mechanism of resistance to this antimalarial. Additionally, mutations within the pfmdr 1 gene have been shown to confer increased resistance to chloroquine, suggesting that they play a role in modulating higher levels of chloroquine resistance.8,9 The same mutations also have been shown to confer quinine resistance and alter the level of resistance and sensitivity to mefloquine and artemisinin.8

The combination of pyrimethamine and sulfadoxine was effective when it was introduced in the late 1960s as a single-dose treatment of acute P. falciparum malaria.10,11 Over the years, however, resistance to Fansidar has been reported, mainly in areas of intense use, particularly areas of chloroquine resistance. Variant sequences of P. falciparum dihydrofolate reductase (DHFR), the target enzyme of pyrimethamine, were first described in 1988.12,13 Resistance to pyrimethamine has been shown to result from a mutation in the DHFR enzyme, changing Ser-108 to Asn-108, and subsequent mutations can greatly increase the level of resistance to this drug.14,15 Ten mutant genotypes for DHFR have been reported from a large number of field samples.4,16–18 Resistance to sulfonamide and sulfones has been shown to result from mutations within dihydropteroate synthetase (DHPS).19,20–22 The amino acid changes at four positions (Ser-436, Gly-437, Ala-581, and Ala-613) have been shown to confer resistance to sulfadoxine and also cross-resistance to sulfones and sulfonamides.21,22 Distribution of mutations in the dhfr and dhps genes and their association with Fansidar resistance have been assessed in different geographic areas, and it has been found that a greater number of mutations in both dhfr and dhps is a good predictor of likely drug failure.4,17,18,23

Vietnam has well-developed systems for malaria surveillance, with recent data showing that more than half the population lives in forests and mountainous areas where they may be at risk of malaria infection. Drug-resistant malaria has been an increasing problem in the last two decades, and although alternative drugs such as artemisinin and its derivatives have become widely used as first-line therapy for malaria, they have been associated with a high frequency of recrudescence.24 In this 1998 study, we have further categorized malaria drug resistance in Vietnam by analyzing mutations in the pfcrt, pfmdr1, dhfr, and dhps genes in P. falciparum primary and recrudescent isolates collected from patients in an area of Binh Phuoc Province highly endemic for malaria.

MATERIALS AND METHODS

Study area and collection of blood samples.

The subjects of the study lived on the Phu Rieng rubber plantation in southern Vietnam. The area is hyperendemic for malaria, which is aggravated by multidrug-resistant P. falciparum. The peak seasons of infection are March–June and September–December (annual main peak of transmission). The annual parasite rate/slide is 10–30% for both P. falciparum and P. vivax.

Blood samples were collected from all P. falciparum-positive cases before administering artemisinin (20 mg/kg on day 0 and 10 mg/kg per day for days 1–4) and following reappearance of parasites after treatment. Blood was drawn by venipuncture into a vacutainer tube containing K-oxalate/Na-fluoride. Thick and thin blood smears were examined after staining with Giemsa.

Parasites, DNA, and drug sensitivity assay.

All parasites were cultivated in vitro by standard methods.25 Nonsynchronized parasites at an initial parasitemia of 0.8–1% were grown at 2% hematocrit in fresh erythrocytes. At the beginning of each experiment, a range of dilutions of each drug, in complete RPMI 1640-Hepes free hypoxanthine medium, 10% serum, and 5% albumax were prepared. Before testing for pyrimethamine susceptibility, parasites were grown in medium containing folate at 0.01 mg/L and p-aminobenzoic acid at 0.5 μg/L without hypoxanthine, supplemented with 8% NaHCO3, 5% albumax, and 10% human serum for 6 days. Drug susceptibility tests were performed using [3H] hypoxanthine uptake as described previously.26 IC50 values were determined from the drug concentration at which [3H] hypoxanthine uptake was reduced by 50% compared with controls. Each assay was performed in triplicate in two independent assays. In parallel with each isolate, standard controls (clones D10 and K1) were tested to establish that no undesired variation in media or drug solutions had occurred. The following drug preparations were used: Chloroquine diphosphate salt (Sigma, Germany), mefloquine hydrochloride (gift from Hoffman LaRoche, Switzerland), quinine hydrochloride (Sigma, Germany), artemisinin (Institute of Quality Control, Hanoi, Vietnam) and pyrimethamine (Sigma).

Sequences of the primers used for detection of polymorphisms in pfmdr1,27,28 pfcrt,7 dhfr, dhps,27,28 and msp229 genes have been described previously.

The pfmdr1 gene was amplified by polymerase chain reaction (PCR) under the following conditions: 1 μL of DNA was used as template, 20–50 pmol of each primer, PCR buffer (50 mM KCL, 10 mM Tris-HCL pH 8.3, 2.5 mM MgCL2), 200 μM dNTPs, 1 unit of Taq polymerase DNA (Perkin-Elmer Cetus, USA) in a final volume of 50 μL. The PCR was performed for 35 cycles at 94°C for 5 seconds, 48°C for 10 seconds, and 68°C for 30 seconds.

Mutations in pfcrt were detected as described previously.7 Amplification of the dhfr gene was by PCR using a reaction volume of 50 μL containing 5 μL of sample DNA, 25 pmoles of each primer, 200 μM dNTPs, 2.5 mM Mgc12, 2.5 U of Taq polymerase (Perkin-Elmer Cetus), PCR buffer (50 mM KCL, 10 mM Tris-HCL, pH 8.3). The PCR assay was performed for 35 cycles using thermocycling conditions (Perkin-Elmer Cetus), which include denaturation at 94°C for 1 min, annealing at 55°C for 1 minute, and extension at 72°C for 45 seconds. The dhps gene was amplified using PCR as described previously.27,28

PCR genotyping of the msp2 gene was performed as described previously.29 If an identified genotype pattern was found in both sample A (primary isolates) and sample B (recurrent isolate), the infection was most likely a recrudescence. In contrast, if the patterns of both samples differed, a reinfection was assumed although a minor population of the original infection may have been responsible.

Restriction fragment length polymorphisms were detected by digesting DNA fragments of pfcrt, pfmdr1, dhfr, and dhps obtained by PCR with the appropriate restriction endonuclease to detect specific point mutations as described previously.17,27,30–33 Digested products were separated on 1.5–3% agarose gel, then stained with ethidium bromide and visualized under UV.

In data analysis, two independent groups were compared using Student’s t test. Mutation points were correlated with drug resistance phenotypes by Fisher’s exact test.

RESULTS

Samples were collected from patients who were positive for P. falciparum before treatment with artemisinin. These primary parasites lines were adapted for in vitro culture to allow detailed analysis of drug resistance phenotypes and genotypes. Additionally, patients who showed breakthrough parasitemias of P. falciparum had samples collected for culture. Of these secondary parasites, 18 were successfully adapted for in vitro culture. DNA was isolated from the culture-adapted parasite lines for analysis of mutations in pfcrt, pfmdr1, dhfr, and dhps genes by PCR. To determine whether there were correlations between the relevant genotypes and drug resistance phenotypes, the same parasite lines also were tested for drug susceptibility to artemisinin, mefloquine, chloroquine, quinine, and pyrimethamine.8,26

Artemisinin, mefloquine, chloroquine, and quinine susceptibility of the primary and secondary isolates.

Nineteen primary P. falciparum lines isolated before drug treatment were adapted for in vitro culture and analyzed for artemisinin, mefloquine, chloroquine, and quinine susceptibility (Table 1 and Figure 1).8,26 These isolates had a mean IC50 for artemisinin of 22.7 nmol (±1.5, 95% confidence intervals, C.I.) and for mefloquine it was 22 nmol (±1.6, 95% C. I.) with 12 of the isolates being classified as resistant to this drug. Eleven primary isolates were resistant to chloroquine; together, they had a mean IC50 of 160.1 nmol (±21.3, 95% C.I.). All but two of the isolates were sensitive to quinine, with a mean IC50 of 286 nmol (±17.5, 95% C.I.).

The secondary P. falciparum isolates derived from patients who developed a parasitemia after artemisinin treatment also were analyzed for susceptibility to artemisinin, mefloquine, chloroquine, and quinine (Table 2 and Figure 1). These secondary isolates had an IC50 for artemisinin of 22.5 nmol (±1, 95% C.I.), which was not significantly different from that of the primary isolates, suggesting that there was no apparent selection for decreased sensitivity to artemisinin with drug treatment. However, these isolates had a higher IC50 for mefloquine of 31.4 nmol (±1.7, 95% C.I.); analysis by paired t test showed this to be highly significant (P = 0.027), suggesting that some selection had occurred for increasing mefloquine resistance because of treatment with artemisinin. The secondary isolates had a lower IC50 for chloroquine, 121.5 nmol (±21.5, 95% C.I.) compared with 160.1 nmol (±21.3 95% C.I.) for the primary isolates. Although this difference was not statistically significant, it was interesting to note that the number of chloroquine-resistant isolates (those with an IC50 greater than 100 nM) was significantly reduced after treatment, suggesting that the decrease in chloroquine resistance was linked to the observed increase in mefloquine resistance of the secondary isolates.8 No significant difference was observed when comparing the mean IC50 for quinine of the primary, 286 nmol (±17.5, 95% C.I.), and secondary, 251 nmol (±16.3, 95% C.I.), isolates. All the isolates except two were found to be highly sensitive to quinine.

The primary and secondary isolates were analyzed to determine whether the parasites isolated after artemisinin treatment represented a breakthrough or a new infection (Table 3). The msp2 gene of each isolate was analyzed by PCR and the allele type classified as different (D) or the same size (S) as judged by mobility of the DNA fragment on agarose gels (Table 3). The parasites isolated after artemisinin treatment that had different msp2 alleles must result from either a new infection or, alternatively, a breakthrough of a minor parasite population from the original infection. The parasite isolates that had an msp2 allele of the same size are likely to represent a breakthrough of the original infection through treatment with artemisinin. Only six of the parasite isolates obtained after artemisinin had msp2 alleles of the same size as the original isolates, suggesting that these were recrudescent infections. Three isolates had mixed msp2 alleles. Nine of the recurrent isolates had different msp2 alleles, which must have originated from new infections or selection of a population from a primary infection with multiple parasites with different msp2 alleles.

The genotypes for pfcrt and pfmdr1 for the primary and secondary isolates.

Genomic DNA from the primary and secondary isolates was analyzed as to the genotype of the pfcrt and pfmdr1 genes to determine whether they were linked to the drug resistance phenotypes.7,8,34,35 There was a perfect correlation with the presence of Tyr-76 encoded by the pfcrt gene in both primary and secondary chloroquine-resistant isolates. Statistical analysis using Fisher’s exact test showed that pfcrt was highly linked to the chloroquine resistance phenotype (P = 0.00002). This is consistent with an important role for the protein encoded by the pfcrt gene in the chloroquine resistance phenotype.7 Of the 12 chloroquine-resistant primary isolates, four encoded Tyr-86 and two had Asp-1042, whereas six had wild-type genotypes in pfmdr1.34,35 Statistical analysis of the presence of mutations in pfmdr1 showed that they also were linked to chloroquine resistance (P = 0.043). These results are consistent with an important role in chloroquine resistance for pfcrt mutations7 and involvement of pfmdr1 in modulation of the level of chloroquine resistance.8 All the secondary isolates had a wild-type sequence for the pfmdr1 gene.34,35

No clear pattern was evident with respect to the genotype of the pfcrt and pfmdr1 genes and susceptibility to quinine and artemisinin in the primary isolates, although susceptibility to mefloquine showed an inverse correlation to chloroquine resistance. This is consistent with previous results, and most isolates that have mutations in the pfmdr1 gene are more susceptible to mefloquine than isolates with a wild-type sequence.8

The dhfr and dhps genotypes of P. falciparum isolates from Vietnam.

To test whether the P. falciparum isolates were multidrug-resistant, we obtained the sequence of the dhfr and dhps genes to determine if they encoded mutations involved in resistance to pyrimethamine and sulfadoxine (Fansidar).12,13,19–22,36,37 Additionally, we determined the sensitivity of the primary parasites for pyrimethamine. Four of the isolates were sensitive to pyrimethamine, and all had Ser-108 whereas all the pyrimethamine-resistant isolates had Asn-108 (Table 4). This is consistent with the essential role of Asn-108 in the mechanism of resistance to this antimalarial. Additionally, the pyrimethamine-resistant isolates had a number of other mutations within dhfr that have been shown previously to be involved in higher levels of resistance.

The sequence of the dhps gene also was obtained for all the isolates to determine whether they encoded mutations associated with resistance to sulfadoxine (Table 4).19,20–22,37 None of the isolates had a wild-type dhps gene, suggesting that there has been strong selection for mutations in this gene. This is consistent with the high levels of pyrimethamine resistance observed in these isolates.

DISCUSSION

Malaria is still a health concern in Vietnam,38 and the spread of drug-resistant P. falciparum has made the selection of appropriate antimalarials for control and treatment problematic. In 1996, the level of P. falciparum chloroquine resistance was approximately 60%, and sulfonamide resistance was 25–40%. The introduction of artemisinin and its widespread availability as a first-line treatment have been important changes in the clinical management of malaria in Vietnam, and the use of artemisinin for treatment and prophylaxis probably has reduced the number of deaths due to malaria.24 The development of decreased sensitivity to artemisinin would threaten its effectiveness. In this study, we have investigated the possibility of selection for decreased artemisinin sensitivity in P. falciparum parasites isolated after patients were treated with artemisinin. No detectable decrease in artemisinin sensitivity was observed. However, there was a statistically significant increase in mefloquine resistance of these isolates after artemisinin treatment failure.

Previous studies of artemisinin sensitivity have shown a wide variation in the level of sensitivity of P. falciparum.39,40 This suggests that there may be genetic alterations that can confer differential sensitivity to artemisinin. Transfection of mutations into the pfmdr1 gene has altered the level of sensitivity to artemisinin, suggesting that the gene is a factor in determining the level of artemisinin sensitivity;8 this agrees with other studies showing that mutations in pfmdr1 confer increased sensitivity to artemisinin in the progeny of a genetic cross of P. falciparum.28 An important finding of these studies was that the altered sensitivity observed with artemisinin also was seen with mefloquine, consistent with a role for pfmdr1 in both of these phenotypes. The observation in this study that the P. falciparum isolates obtained after drug treatment were more mefloquine-resistant suggests that artemisinin treatment has directly selected for this increased drug resistance. This is consistent with the absence of pfmdr1 mutations in all the parasite isolates obtained after artemisinin treatment, whereas six of the primary isolates had mutations in this gene.

If artemisinin treatment selected for increased mefloquine resistance and the absence of mutations in pfmdr1, then why was no statistically significant difference seen for artemisinin? Previously, it has been shown that the measurement of artemisinin sensitivity is highly influenced by the inoculum effect in P. falciparum.41 The differences in artemisinin sensitivity that might be expected between the primary isolates and those isolated after artemisinin treatment would be quite small.39,40 Consequently, it may be difficult to quantitate the differences accurately enough to observe a slight decrease in artemisinin sensitivity. It will be interesting to follow artemisinin sensitivity in Vietnam over time to determine whether decreased sensitivity, and perhaps resistance, to this drug is developing.

It is clear from this study that T-76 in the pfcrt gene is tightly linked to chloroquine resistance, as all chloroquine-resistant isolates had this mutation whereas all chloroquine-sensitive isolates had the wild-type sequence.7 This is consistent with an essential role for pfcrt mutations in P. falciparum resistance to chloroquine. Mutations in the pfmdr1 gene also were linked to chloroquine resistance. However, it is clear that they are not required for resistance, as some chloroquine-resistant isolates had wild-type sequence for this gene. Previous results have suggested that mutations in pfmdr1 play a role in higher levels of chloroquine resistance, and the results presented here are consistent with this.8,9

The increased mefloquine resistance of the secondary isolates was concomitant with a decrease in the level of chloroquine resistance, which has been previously observed in in vitro selected P. falciparum lines.42,43 Selection of parasites for increased mefloquine resistance resulted in a decrease in the level of chloroquine resistance as well as amplification of the pfmdr1 gene. This was consistent with involvement in these phenotypes of increased expression of the protein encoded by pfmdr1. Transfection of different pfmdr1 alleles into P. falciparum also had an inverse effect on resistance for chloroquine compared with that of mefloquine. The observations in this study, that the secondary parasites were more mefloquine- and less chloroquine-resistant and encoded the wild-type pfmdr1 allele, are consistent with direct selection on this gene that alters sensitivity to these drugs. This selection must have occurred during artemisinin treatment.

Although Fansidar was not used for treatment in this study, it was of interest to determine whether these parasites were resistant to multiple drugs. We therefore tested the level of sensitivity to pyrimethamine and the genotype of the dhfr and dhps genes that have previously been shown to contain specific mutations that encode resistance to these drugs.14,15,19,20–22 We did not determine the sensitivity to sulfadoxine because of the difficulty of obtaining accurate measurements for this drug in vitro.44 The presence of Asn-108 in DHFR showed a complete linkage with pyrimethamine resistance, as expected from previous results. Only four isolates were sensitive to pyrimethamine, and all had mutations in the dhps gene, suggesting that there has been strong Fansidar pressure against P. falciparum in this area of Vietnam. The Gly-437 mutation in DHPS was observed in all isolates except one, consistent with this mutation’s being the first in this enzyme as a result of sulfa drug pressure.21,22

The emergence of P. falciparum parasites that are resistant to multiple antimalarials has caused a major dilemma in the selection of suitable drugs for the control and treatment of malaria caused by this parasite. Artemisinin is an important antimalarial that is effective against P. falciparum parasites resistant to drugs such as chloroquine. The increasing identification of parasites that are resistant to many antimalarials, as well as the potential for the development of artemisinin resistance, is a major cause for concern. It is important that the level of artemisinin sensitivity of P. falciparum be monitored in areas such as Vietnam where the drug is extensively used to allow early detection of the development of resistance.

Table 1

The response of Plasmodium falciparum in Vietnam to chloroquine, mefloquine, quinine, and artemisinin

IC50 (nmol)Pfcrt genotypePfmdr1 genotype
IsolatesArtemisininMefloquineChloroquineQuinine7686103410421242
# Threshold of resistance.
* Isolates below this line are defined as chloroquine-resistant.
T6531.3 ± 0.934.7 ± 4.315.4 ± 6.3271.8 ± 20.7KNSND
T1714.7 ± 6.318.6 ± 6.321.4 ± 0.697.7 ± 11.3KNSND
T4412.0 ± 0.728.0 ± 0.228.1 ± 2.7199.7 ± 52.0KNSND
T6031.2 ± 3.630.0 ± 5.328.5 ± 5.8235.9 ± 3.2KNSND
T416.0 ± 1.326.2 ± 3.336.1 ± 0.1321.2 ± 68.8KNSND
T4331.2 ± 0.238.1 ± 0.140.0 ± 3.5398.2 ± 67.3KNSND
*T1613.0 ± 0.123.7 ± 0.542.4 ± 5.0150.1 ± 11.8KNSND
T2238.3 ± 2.715.5 ± 1.2106.1 ± 7.3348.3 ± 0.9TNSND
T5916.2 ± 2.126.2 ± 5.4108.0 ± 1.7338.0 ± 63.6TNSND
T5426.3 ± 0.410.6 ± 0.1131.6 ± 47.4291.8 ± 7.4TYSND
T4035.6 ± 3.623.0 ± 0.6133.4 ± 5.5208.4 ± 4.6TNSND
T2936.1 ± 7.19.2 ± 0.3134.2 ± 20.8214.2 ± 5.7TYSND
T3013.1 ± 2.711.0 ± 0.5183.2 ± 21.5426.2 ± 50.1TNSDD
T2518.6 ± 6.832.3 ± 0.1239.7 ± 12.3289.7 ± 47.6TYSND
T915.4 ± 2.127.2 ± 3.3292.2 ± 2.9145.3 ± 24.8TNSND
T6436.9 ± 2.25.4 ± 0.1335.6 ± 1.6263.5 ± 10.2TYSND
T1515.5 ± 3.65.5 ± 1.1357.8 ± 27.8532.3 ± 2.6TNSND
T314.6 ± 0.319.9 ± 0.4387.1 ± 8.9465.9 ± 22.6TNSDD
T816.0 ± 0.133.1 ± 4.2421.3 ± 14.9236.8 ± 33.2TNSND
TOR#20100450
Table 2

The response of Plasmodium falciparum isolates from recurrent infections to chloroquine, mefloquine, quinine, and artemisinin

IC50 (nmol)Pfcrt genotypePfmdr1 genotype
IsolatesArtemisininMefloquineChloroquineQuinine7686103410421242
#Threshold of resistance.
*Isolates above this line are defined as chloroquine-resistant.
T3-R2111.7 ± 0.728.9 ± 0.914.5 ± 0.1285.6 ± 43.6KNSND
T17-R1415.6 ± 0.814.2 ± 0.420.0 ± 1.760.2 ± 1.7KNSND
T8-R2120.5 ± 1.725.0 ± 0.821.2 ± 0.7124.6 ± 4.5KNSND
T4-R1911.7 ± 1.436.9 ± 4.521.2 ± 1.0321.2 ± 68.9KNSND
T43-R2523.9 ± 1.532.4 ± 2.631.0 ± 0.7183.5 ± 15.7KNSND
T64-R1818.4 ± 1.049.3 ± 3.933.6 ± 4.6196.3 ± 8.3KNSND
T40-R1725.3 ± 1.432.9 ± 5.237.4 ± 9.2138.6 ± 9.9KNSND
T54-2223.2 ± 0.738.4 ± 3.038.4 ± 0.3298.7 ± 7.7KNSND
T60-R1736.9 ± 2.253.3 ± 5.940.6 ± 3.6300.2 ± 24.2KNSND
T44-R1424.0 ± 1.940.3 ± 3.745.7 ± 2.4133.0 ± 25.6KNSND
T29-R1818.7 ± 1.536.4 ± 8.948.0 ± 7.5221.4 ± 23.2KNSND
*T16-R1828.9 ± 0.737.0 ± 3.450.6 ± 3.0245.7 ± 17.7KNSND
T25-R2619.1 ± 0.521.6 ± 2.0149.8 ± 4.9301.2 ± 47.7TNSND
T9-R2121.4 ± 1.721.5 ± 4.2161.2 ± 21.5289.0 ± 75.0TNSND
T65-R2121.9 ± 0.429.1 ± 4.1163.6 ± 4.0278.6 ± 14.9TNSND
T22-R2432.4 ± 2.133.5 ± 1.4193.2 ± 35.9313.1 ± 6.2TNSND
T59-R1826.5 ± 3.417.7 ± 0.7310.0 ± 6.1318.1 ± 22.0TNSND
T15-R1420.7 ± 1.016.6 ± 5.4420.7 ± 30.4511.8 ± 5.9TNSND
TOR#30100450
Table 3

Comparison of artemisinin and mefloquine IC50 for primary and recrudescent parasite isolates

IC50 Artemisinin*IC50 Mefloquine*
1°-Rc# IsolatesRcRcMSP2 allele@
* IC50 for artemisinin and mefloquine are in nmol.
# The isolates listed include the primary parasites and recrudescent parasites (Rc).
@ D = different size; S = same size.
T64-R1836.9 ± 2.218.4 ± 1.05.4 ± 0.149.3 ± 3.9D
T15-R1415.5 ± 2.120.7 ± 1.05.5 ± 1.016.6 ± 5.4S
T29-R1836.1 ± 7.118.7 ± 1.59.2 ± 0.336.4 ± 8.9D
T54-R2226.3 ± 0.423.2 ± 0.710.7 ± 0.138.4 ± 3.0D
T22-R2438.3 ± 2.732.4 ± 2.115.5 ± 1.233.5 ± 1.4D
T17-R1414.7 ± 6.315.6 ± 0.818.6 ± 6.314.2 ± 0.4D
T3-R2114.6 ± 0.311.7 ± 0.719.9 ± 0.428.9 ± 0.9D
T16-R1813.0 ± 0.128.8 ± 0.723.7 ± 0.537.0 ± 3.4D
T59-R1816.2 ± 2.126.6 ± 3.426.2 ± 5.417.7 ± 0.7S
T4-R1916.0 ± 1.311.7 ± 1.426.2 ± 3.336.9 ± 4.5S
T9-R2115.4 ± 2.121.4 ± 1.727.2 ± 3.321.5 ± 4.2S
T40-R1728.3 ± 1.225.3 ± 1.423.1 ± 0.632.9 ± 5.2S + D
T44-R1412.0 ± 0.724.0 ± 1.928.0 ± 0.240.3 ± 3.7S + D
T60-R1731.2 ± 3.636.9 ± 2.230.1 ± 5.353.3 ± 5.9S
T25-R2618.6 ± 6.819.1 ± 0.532.3 ± 0.121.6 ± 2.0D
T8-R2116.0 ± 0.120.5 ± 1.733.1 ± 4.225.1 ± 0.8D
T65-R2131.3 ± 0.921.9 ± 0.434.7 ± 4.329.1 ± 4.1S + D
T43-R2531.2 ± 0.223.9 ± 1.538.1 ± 0.132.4 ± 2.6S
Table 4

Response of Plasmodium falciparum isolates to pyrimethamine compared with their dhfr and dhps genotypes

dhfr genotype#dhps genotype*
IsolatesPyrimethamine IC50 (μg/mL)165159108164436437540581613
#The amino acid positions within dhfr associated with pyrimethamine resistance.
* The amino acid positions in dhps associated with sulfadoxine resistance (Triglia et al., 1998).
@ Isolates above this line are defined as pyrimethamine sensitive while those below are resistant.
T430.006ANCSIFGKAA
T600.008AIRSISGKAA
T650.013AICSISGKAA
@ T440.022ANCSIFGKAA
T41.63ANRNISGEAS
T175.25AIRNISGKAA
T546.31AIRNISAKGA
T226.33AIRNISGKGA
T407.50AIRNLSGEAS
T308.61AIRNLSGEAS
T99.33AIRNLSGEGA
T159.57AIRNLSGEAS
T1610.92ANRNLFGKAA
T2915.95AIRNISGKGA
T6416.29AIRNISGKGA
T2518.61AIRNISGEAA
T5921.76AIRNLSGEAA
T325.68AIRNLSGEAS
T828.96AIRNIFGEGA
Fig. 1.
Fig. 1.

A plot of the IC50 for primary and recrudescent isolates for artemisinin (A), mefloquine (B), chloroquine (C) and quinine (D). Each dot corresponds to the IC50 of a particular isolate and the line signifies the mean of the isolates. The arrow in B, C and D refers to the threshold of resistance obtained previously by determining serum drug concentration in patients infected with sensitive and resistant forms of P. falciparum.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 3; 10.4269/ajtmh.2003.68.350

Authors’ addresses: Thanh Ngo, National Institute of Malariology, Parasitology, and Entomology, Luong The Vinh Road, Hanoi, Vietnam. Manoj Duraisingh and Alan F. Cowman, Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia. Michael Reed. Tuberculosis Research Section, NIAID, Twinbrook 11, Room 235, 12441 Parklawn Drive, Rockville, MD 20852. David Hipgrave, University of Melbourne, 57 Quang Trung, Level 5, Hanoi, Vietnam. Beverley Biggs, University of Melbourne, Parkville, Victoria 3050, Australia.

Acknowledgments: We thank David Warhurst for providing information before publication that allowed us to genotype the pfmdr1, dhfr, and dhps genes. We would like to acknowledge the Australian Red Cross Blood Bank in Melbourne for supplying human erythrocytes and serum, and the Australian Agency for International Development for support of the field work. AFC is an International Research Scholar of the Howard Hughes Medical Institute.

Financial support: This work is supported by a grant from the National Health and Medical Research Council of Australia.

REFERENCES

  • 1

    WHO, 1997. Malaria in the South-East Asia Region, Regional office for South-East Asia, New Delhi.

  • 2

    Phan VT, 1990. Technical problems and solution of malaria eradication in Vietnam. Vietnamese scientific research report period 1986–1990 (NIMPE, Vietnam). 9–21.

  • 3

    Cong LD, Sy ND, Hinh TD, Huong NV, Thanh NV, Tien NT, Van Manh DH, 1994. Study on appropriate solutions for control of malaria parasite drug - resistance and vector prevention. Information for the control and prevention of malaria in Vietnam. 2 :1–5.

    • Search Google Scholar
    • Export Citation
  • 4

    Zindrou S, Dung NP, Sy ND, Skold O, Swedberg G, 1996. Plasmodium falciparum: mutation pattern in the dihydrofolate reductase-thymidylate synthase genes of Vietnamese isolates, a novel mutation, and coexistence of two clones in a Thai patient. Exp Parasitol 84 :56–64.

    • Search Google Scholar
    • Export Citation
  • 5

    Am NT, 1993. Malaria in Vietnam—environment, prevention and treatment. Bull Soc Pathol Exot 86 :494–499.

  • 6

    Krogstad DJ, Gluzman IY, Kyle DE, Oduola AM, Martin SK, 1987. Efflux of chloroquine from Plasmodium falciparum: mechanism of chloroquine resistance. Science 238 :1283–1285.

    • Search Google Scholar
    • Export Citation
  • 7

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

    • Search Google Scholar
    • Export Citation
  • 8

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

    • Search Google Scholar
    • Export Citation
  • 9

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

    • Search Google Scholar
    • Export Citation
  • 10

    Harinasuta T, Viravan C, Reid HA, 1967. Sulphormethoxine in chloroquine-resistant falciparum malaria in Thailand. Lancet 1 :1117–1119.

  • 11

    Harinasuta T, Viravan C, Buranasin P, 1988. Parenteral Fansidar in falciparum malaria. Trans R Soc Trop Med Hyg 82 :694.

  • 12

    Cowman AF, Morry MJ, Biggs BA, Cross GAM, Foote SJ, 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci U S A 85 :9109–9113.

    • Search Google Scholar
    • Export Citation
  • 13

    Peterson DS, Walliker D, Wellems TE, 1988. Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc Natl Acad Sci U S A 85 :9114–9118.

    • Search Google Scholar
    • Export Citation
  • 14

    Sirawaraporn W, Yuthavong Y, 1984. Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi. Mol Biochem Parasitol 10 :355–367.

    • Search Google Scholar
    • Export Citation
  • 15

    Wu Y, Kirkman LA, Wellems TE, 1996. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc Natl Acad Sci U S A 93 :1130–1134.

    • Search Google Scholar
    • Export Citation
  • 16

    Basco LK, de Pecoulas PE, Wilson CM, Le Bras J, Mazabraud A, 1995. Point mutations in the dihydrofolate reductase-thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum. Mol Biochem Parasitol 69 :135–138.

    • Search Google Scholar
    • Export Citation
  • 17

    Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, Winstanley PA, Estrada-Franco JG, Mollinedo RE, Avila JC, Cespedes JL, Carter D, Doumbo OK, 1997. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance. J Infect Dis 176 :1590–1596.

    • Search Google Scholar
    • Export Citation
  • 18

    Nagesha HS, Din-Syafruddin D, Casey GJ, Susanti AI, Fryauff DJ, Reeder JC, Cowman AF, 2001. Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in vivo drug resistance in West Papua, Indonesia. Trans R Soc Trop Med Hyg 95 :1–7.

    • Search Google Scholar
    • Export Citation
  • 19

    Triglia T, Cowman AF, 1994. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc Natl Acad Sci U S A 91 :7149–7153.

    • Search Google Scholar
    • Export Citation
  • 20

    Brooks D, Wang P, Read M, Watkin W, Sims P, Hyde J, 1994. Sequence variation in the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine. Eur J Biochem 224 :397–405.

    • Search Google Scholar
    • Export Citation
  • 21

    Triglia T, Menting JGT, Wilson C, Cowman AF, 1997. Mutations of dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A 94 :13944–13949.

    • Search Google Scholar
    • Export Citation
  • 22

    Triglia T, Wang P, Sims PFG, Hyde JE, Cowman AF, 1998. Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria. EMBO J 17 :3807–3815.

    • Search Google Scholar
    • Export Citation
  • 23

    Wang P, Read M, Sims PF, Hyde JE, 1997. Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization. Mol Microbiol 23 :979–986.

    • Search Google Scholar
    • Export Citation
  • 24

    Hien TT, VinhChau NV, Vinh NT, Hunh NT, Phung MY, Toan LM, Mai PP, Dung NT, HoaiTam DT, Arnold K, 1997. Management of multiple drug-resistant malaria in Vietnam. Ann Acad Med 26 :659–663.

    • Search Google Scholar
    • Export Citation
  • 25

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193 :673–675.

  • 26

    Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16 :710–718.

    • Search Google Scholar
    • Export Citation
  • 27

    Duraisingh MT, Curtis J, Warhurst DC, 1998. Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp Parasitol 89 :1–8.

    • Search Google Scholar
    • Export Citation
  • 28

    Duraisingh MT, Roper C, Walliker D, Warhurst DC, 2000. Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum. Mol Microbiol 36 :955–961.

    • Search Google Scholar
    • Export Citation
  • 29

    Felger I, Tavul L, Beck H-P, 1993. Plasmodium falciparum: a rapid technique for genotyping the merozoite surface protein 2. Exp Parasitol 77 :372–375.

    • Search Google Scholar
    • Export Citation
  • 30

    Duraisingh MT, Drakeley CJ, Muller O, Bailey R, Snounou G, Targett GA, Greenwood BM, Warhurst DC, 1997. Evidence for selection for the tyrosine-86 allele of the pfmdr 1 gene of Plasmodium falciparum by chloroquine and amodiaquine. Parasitology 114 :205–211.

    • Search Google Scholar
    • Export Citation
  • 31

    Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE, 1995. Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52 :565–568.

    • Search Google Scholar
    • Export Citation
  • 32

    Plowe CV, Djimde A, Wellems TE, Diop S, Kouriba B, Doumbo OK, 1996. Community pyrimethamine-sulfadoxine use and prevalence of resistant Plasmodium falciparum genotypes in Mali: a model for deterring resistance. Am J Trop Med Hyg 55 :467–471.

    • Search Google Scholar
    • Export Citation
  • 33

    Plowe CV, Kublin JG, 1998. P. falciparum DHFR and DHPS mutations: epidemiology and role in clinical resistance to antifolates. Drug Resistance Updates.

  • 34

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

    • Search Google Scholar
    • Export Citation
  • 35

    Foote SJ, Thompson JK, Cowman AF, Kemp DJ, 1989. Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum. Cell 57 :921–930.

    • Search Google Scholar
    • Export Citation
  • 36

    Sirawaraporn W, Sirawaraporn R, Cowman AF, Yuthavong Y, Santi DV, 1990. Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum. Biochemistry 29 :10779–10785.

    • Search Google Scholar
    • Export Citation
  • 37

    Wang P, Read M, Sims PFG, Hyde JE, 1997. Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilisation. Mol Microbiol 23 :979–986.

    • Search Google Scholar
    • Export Citation
  • 38

    Morillon M, Baudon D, Dai B, 1996. Malaria in Vietnam in 1996: brief synthesis of epidemiological data. Med Trop 56 :197–200.

  • 39

    Basco LK, Le Bras J, 1993. In vitro activity of artemisinin derivatives against African isolates and clones of Plasmodium falciparum. Am J Trop Med Hyg 49 :301–307.

    • Search Google Scholar
    • Export Citation
  • 40

    Pradines B, Rogier C, Fusai T, Tall A, Trape JF, Doury JC, 1998. In vitro activity of artemether against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial drugs. Am J Trop Med Hyg 58 :354–357.

    • Search Google Scholar
    • Export Citation
  • 41

    Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 1999. Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study. Parasitology 119 :435–440.

    • Search Google Scholar
    • Export Citation
  • 42

    Barnes DA, Foote SJ, Galatis D, Kemp DJ, Cowman AF, 1992. Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum. EMBO J 11 :3067–3075.

    • Search Google Scholar
    • Export Citation
  • 43

    Peel SA, Bright P, Yount B, Handy J, Baric RS, 1994. A strong association between mefloquine and halofantrine resistance and amplification, overexpression, and mutation in the Pglycoprotein gene homolog (pfmdr) of Plasmodium falciparum in vitro. Am J Trop Med Hyg 51 :648–658.

    • Search Google Scholar
    • Export Citation
  • 44

    Wang P, Sims PFG, Hyde JE, 1997. A modified in vitro sulfadoxine susceptibility assay for Plasmodium falciparum suitable for investigating Fansidar resistance. Parasitology 115 :223–230.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Alan F. Cowman, Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Melbourne, 3050, Australia, Telephone: 62-3-93452555, Fax: 62-3-93470852, E-mail: cowman@wehi.edu.au
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