• 1.

    World Health Organization, 2005. Susceptibility of Plasmodium falciparum to Antimalarial Drugs. Report on Global Monitoring 1996–2004: Geneva: World Health Organization.

    • Search Google Scholar
    • Export Citation
  • 2.

    Dondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, von Seidlein L, 2010. Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol 8: 272280.

    • Search Google Scholar
    • Export Citation
  • 3.

    Krishna S, Pulcini S, Fatih F, Staines H, 2010. Artemisinins and the biological basis for the PfATP6/SERCA hypothesis. Trends Parasitol 26: 517523.

    • Search Google Scholar
    • Export Citation
  • 4.

    Uhlemann AC, Cameron A, Eckstein-Ludwig U, Fischbarg J, Iserovich P, Zuniga FA, East M, Lee A, Brady L, Haynes RK, Krishna S, 2005. A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nat Struct Mol Biol 12: 628629.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O, 2005. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 366: 19601963.

    • Search Google Scholar
    • Export Citation
  • 6.

    Witkowski B, Lelièvre J, Lopez Barragan MJ, Laurent V, Su XZ, Berry A, Benoit-Vical F, 2010. Increased tolerance to artemisinin in Plasmodium falciparum is mediated by a quiescence mechanism. Antimicrob Agents Chemother 54: 18721877.

    • Search Google Scholar
    • Export Citation
  • 7.

    Pearce RJ, Pota H, Evehe MSB, Ba EH, Mombo-Ngoma G, Malisa AL, Ord R, Inojosa W, Matondo A, Diallo DA, Mbacham W, van den Broek IV, Swarthout TD, Getachew A, Dejene S, Grobusch MP, Njie F, Dunyo S, Kweku M, Owusu-Agyei S, Chandramohan D, Bonnet M, Guthmann JP, Clarke S, Barnes KI, Streat E, Katokele ST, Uusiku P, Agboghoroma CO, Elegba OY, Cisse B, A-Elbasit IE, Giha HA, Kachur SP, Lynch C, Rwakimari JB, Chanda P, Hawela M, Sharp B, Naidoo I, Roper C, 2009. Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria. PLoS Med 6: e1000055.

    • Search Google Scholar
    • Export Citation
  • 8.

    Whegang SY, Tahar R, Foumane VN, Soula G, Gwet H, Thalabard JC, Basco LK, 2010. Efficacy of non-artemisinin- and artemisinin-based combination therapies for uncomplicated malaria in Cameroon. Malar J 9: 56. Available at: http://www.malariajournal.com/content/9/1/56.

    • Search Google Scholar
    • Export Citation
  • 9.

    World Health Organization, 2003. Assessment and Monitoring of Antimalarial Drug Efficacy for the Treatment of Uncomplicated falciparum Malaria. Geneva: World Health Organization, WHO/HTM/RBM/2003.50.

    • Search Google Scholar
    • Export Citation
  • 10.

    World Health Organization, 2008. Methods and Techniques for Clinical Trials on Antimalarial Drug Efficacy: Genotyping to Identify Parasite Populations. Informal Consultation Organized by the Medicines for Malaria Venture and Cosponsored by the World Health Organization, May 29–31, 2007, Amsterdam, The Netherlands.

    • Search Google Scholar
    • Export Citation
  • 11.

    Eldin de Pécoulas P, Basco LK, Abdallah B, Djé MK, Le Bras J, Mazabraud A, 1995. Plasmodium falciparum: detection of antifolate resistance by mutation-specific restriction enzyme digestion. Exp Parasitol 80: 483487.

    • Search Google Scholar
    • Export Citation
  • 12.

    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: 18.

    • Search Google Scholar
    • Export Citation
  • 13.

    Tahar R, Ringwald P, Basco LK, 2009. Molecular epidemiology of malaria in Cameroon. XXVIII. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum and sequence analysis of the P. falciparum ATPase 6 gene. Am J Trop Med Hyg 81: 1318.

    • Search Google Scholar
    • Export Citation
  • 14.

    Tahar R, Basco LK, 2007. Molecular epidemiology of malaria in Cameroon. XXVII. Clinical and parasitological response to sulfadoxine-pyrimethamine treatment and Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase alleles in Cameroonian children. Acta Trop 103: 8189.

    • Search Google Scholar
    • Export Citation
  • 15.

    Mugittu K, Genton B, Mshinda H, Beck HP, 2006. Molecular monitoring of Plasmodium falciparum resistance to artemisinin in Tanzania. Malar J 5: 126. Available at: http://www.malariajournal.com/content/5/1/126.

    • Search Google Scholar
    • Export Citation
  • 16.

    Dahlström S, Veiga MI, Ferreira P, Mårtensson A, Kaneko A, Andersson B, Björkman A, Gil JP, 2008. Diversity of the sarco/endoplasmic reticulum Ca2+-ATPase orthologue of Plasmodium falciparum (PfATP6). Infect Genet Evol 8: 340345.

    • Search Google Scholar
    • Export Citation
  • 17.

    Menegon M, Sannella AR, Majori G, Severini C, 2008. Detection of novel point mutations in the Plasmodium falciparum ATPase 6 candidate gene for resistance to artemisinins. Parasitol Int 57: 233235.

    • Search Google Scholar
    • Export Citation
  • 18.

    Zhang GQ, Guan Y, Zheng B, Wu S, Tang LH, 2008. No PfATPase6 S769N mutation found in Plasmodium falciparum isolates from China. Malar J 7: 122. Available at: http://www.malariajournal.com/content/7/1/112.

    • Search Google Scholar
    • Export Citation
  • 19.

    Ibrahim ML, Khim N, Adam HH, Ariey F, Duchemin JB, 2009. Polymorphism of PfATPase in Niger: detection of three new point mutations. Malar J 8: 28. Available at: http://www.malariajournal.com/content/8/1/28.

    • Search Google Scholar
    • Export Citation
  • 20.

    Cojean S, Hubert V, Le Bras J, Durand R, 2006. Resistance to dihydroartemisinin. Emerg Infect Dis 12: 17981799.

 

 

 

 

Molecular Epidemiology of Malaria in Cameroon. XXX. Sequence Analysis of Plasmodium falciparum ATPase 6, Dihydrofolate Reductase, and Dihydropteroate Synthase Resistance Markers in Clinical Isolates from Children Treated with an Artesunate-Sulfadoxine-Pyrimethamine Combination

View More View Less
  • Organisation de Coordination pour la Lutte Contre les Endémies en Afrique Centrale, Institut de Recherche pour le Développement, Yaoundé, Cameroon; Département de Biologie Animale et Physiologie, Université de Yaoundé I, Yaoundé, Cameroon; Unité Mixte de Recherche 216, Institut de Recherche pour le Développement, Université René Descartes-Paris V, Paris, France; Unité Mixte de Recherche 198, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Institut de Recherche pour le Développement, Université Aix-Marseille 2, Marseille, France

Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance.

Introduction

Plasmodium falciparum malaria parasite has become resistant to most affordable antimalarial drugs, such as chloroquine (CQ), amodiaquine (AQ), and sulfadoxine-pyrimethamine (SP).1 To circumvent the problem of drug resistance, national health authorities of many concerned countries, with the support of the World Health Organization, have resorted to the use of artemisinin-based combination therapies (ACTs) for the first-line treatment of uncomplicated malaria. However, a decrease in the sensitivity to artemisinin has been recently documented in Cambodia, necessitating an increased vigilance in monitoring drug-resistant malaria.2

The mechanism of action of artemisinin is not well understood. One of the hypotheses is based on the specific inhibition of P. falciparum ATPase 6 (PfATP6), an orthologue gene product of the mammal sarco-endoplasmic reticulum calcium-dependent ATPase (SERCA).3 Initial laboratory studies have suggested that L263E substitution in PfATP6 affects the active site and induces conformational changes, reducing the affinity between the enzyme and artemisinin.4 Subsequent studies carried out on field isolates have shown an association between increased 50% inhibitory concentration (IC50) for artemether and either a single amino acid substitution S769N (in South American strains) or double amino acid substitutions E431K and A623E (in African strains).5 Quiescence is another possible mechanism of resistance demonstrated in laboratory-adapted P. falciparum. Ring stages of artemisinin-tolerant P. falciparum strain may undergo quiescence, i.e. developmental arrest, during exposure in vitro to high concentrations of artemisinin derivatives, and pursue normal process of cell cycle once the drug is removed.6

The mechanisms of resistance to sulfadoxine and pyrimethmine have been extensively studied. The key amino acid substitutions associated with in vitro resistance to sulfadoxine and pyrimethamine are Ala437Gly and Ser108Asn, respectively. However, for clinical resistance to occur, additional mutations, commonly referred to as quintuple dihydrofolate reductase (dhfr)–dihydropteroate synthase (dhps) mutations, are required.7

In this study, we assessed the mutations of dhfr, dhps, and P. falciparum ATPase 6 (pfatp6) genes associated with drug resistance in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine (AS-SP) combination and followed-up for 28 days. We also assessed the usefulness of molecular markers as a complementary tool for the evaluation of therapeutic efficacy of ACT. The efficacy of AS-SP, in relation to other ACTs, has been analyzed in our previous study.8

Materials and Methods

Patients and blood collection.

Blood samples were obtained during February–May 2005 as part of a randomized clinical study that compared the therapeutic efficiency of amodiaquine monotherapy, artesunate-amodiaquine, and AS-SP combinations.8 Fingerprick capillary blood was collected on Isocode Stix® filter papers (Schleicher and Schuell, Ecquevilly, France) from children less than five years of age who came to the Nlongkak Catholic Missionary Dispensary in Yaoundé, Cameroon. Children were enrolled in the study if they satisfied the following criteria set by the World Health Organization: presence of P. falciparum with a parasite density > 2,000 asexual parasites/μL of blood without any other Plasmodium species, fever > 37.5°C, hematocrit > 15%, absence of severe malnutrition and other infectious diseases that may be the origin of fever, absence of signs and symptoms of severe and complicated malaria, and a signed written informed consent provided by parents or a legal guardian.9

Patients were treated with artesunate at a dose of 4 mg/kg of body weight administered per day on days 0, 1, and 2. The standard dose of SP (25 mg/kg of body weight for sulfadoxine and 1.25 mg/kg of body weight for pyrimethamine) was administered in a single dose. Patients were followed-up on days 1, 2, 3, 7, 14, 21, and 28, as recommended in the 2003 World Health Organization protocol.9 Each dose of antimalarial drugs was administered under supervision during the visits. Patients who failed to respond to the assigned drug were treated with oral quinine (25 mg/kg of body weight/day for 5 days) or the standard dose of artemether-lumefantrine, one of the first-line ACTs in Cameroon. In case of treatment failure, the polymorphic merozoite surface antigen-1, merozoite surface antigen-2, and glutamine-rich protein genes of the pre-treatment and recrudescent samples were compared by agarose gel electrophoresis to distinguish between recrudescence and reinfection, as recommended by a group of malaria experts.10

DNA extraction and polymerase chain reaction.

Parasite DNA was extracted from filter paper by the boiling method as recommended by the manufacturer of the Isocode Stix® filter papers (Schleicher and Schuell). Briefly, after rinsing the filter paper once in 500 μL of sterile distilled water, the filter paper was placed into a 0.5-mL microtube into which 75 μL of sterile distilled water was added. The filter paper was incubated at 100°C for 20 minutes and agitated for a few seconds. Ten microliters of the supernatant was used to amplify pfdhfr, pfdhps, and pfatp6 gene fragments.

The pfdhfr mutations at codons 51, 59, 108, and 164 and pfdhps mutations at codons 436, 437, and 540 were determined by nested polymerase chain reaction, followed by enzymatic digestion, as described by Eldin de Pécoulas and others11 and Duraisingh and others.12 The DHFR amino acid residue 16 and DHPS amino acid residues 581 and 613 were not analyzed in this study because mutations at these positions are rare in African isolates and the Ala16Val substitution in DHFR occurs with the rare Ser108Thr substitution.7

For pfatp6 mutation analysis, a gene fragment of 1,793 basepairs corresponding to exon 1 of the coding region was amplified by the nested polymerase chain reaction (PCR) protocol described in our previous study.13 Amplification products were sequenced from the 5′- and 3′-ends by using an automated DNA sequencer (ABI Prism; Perkin Elmer Corp., Les Ulis, France) to determine the codons that were reported to be associated with artemisinin resistance (amino acid residues 263, 431, 623, and 769) and possible novel mutations in the pfatp6 gene.4,5 On the basis of these studies, the wild-type haplotype was defined as LEAS. Known pfatp6 mutants include the West African double mutant type (E431K + A623E) and the South American single mutant type (S769N).

Statistical analysis.

Fisher's exact test was used to analyze the relationship between the clinical outcome of AS-SP treatment and sequence polymorphisms of drug resistance markers. The significance level was fixed at 0.05.

Results

Sixty-one isolates obtained on day 0 from all patients treated with AS-SP combination were included in the present study. The pfdhfr and pfdhps fragments of all 61 isolates were successfully amplified. All 61 isolates were triple mutants, i.e., carried mutant 51, 59, and 108 dhfr alleles (Asn51Ile, Cys59Arg, Ser108Asn; haplotype IRN). On the basis of results of dhfr and dhps sequences, 42 (69%) isolates were classified as quadruple mutants, i.e., triple dhfr mutation (Asn51Ile, Cys59Arg, Ser108Asn) and Ala437Gly dhps mutation. There was no quintuple mutant (quadruple mutation + dhps mutant allele Lys540Gln). Mutation was not detected at DHFR-164.

On the basis of amino acid substitutions at positions 263, 431, 623, and 769 that have been linked to artemisinin resistance in previous studies,4,5 the mutant haplotype LKAS was observed in 18% of isolates. The E432K mutation was present as a pure allele in two isolates. Two non-synonymous pfatp6 mutations (E641Q, n = 3 isolates and E643Q, n = 1 isolate) and three synonymous mutations (440, 594, and 621 in three different isolates) were also found. The pfatp6 haplotypes (positions 263, 431, 432, 623, and 769) are summarized in Table 1. Because of the low rate of pfatp6 mutations among 61 isolates obtained from patients treated with AS-SP and that pure or mixed E431K allele occurred only in patients with an adequate clinical and parasitologic response outcome, possible associations between molecular markers and clinical outcome were not analyzed.

Table 1

Plasmodium falciparum dhfr, pfdhps, and pfatp6 sequence polymorphisms of isolates and clinical response of children treated with artesunate-sulfadoxine-pyrimethamine combination, Cameroon*

No. of isolates (%)DHFRDHPSPFATP6Outcome
5 (8.2)IRNSALEEASACPR
1 (1.6)IRNSALEEASLPF
7 (11.5)IRNA/FALEEASACPR
3 (4.9)IRNA/FALKEASACPR
2 (3.3)IRNS + A/FALEEASACPR
1 (1.6)IRNA/FALEEASLPF (ACPR)
1 (1.6)IRNSA/GLEEASACPR
1 (1.6)IRNSA/GLKEASACPR
13 (21.3)IRNSGLEEASACPR
1 (1.6)IRNSGLEEASLPF
1 (1.6)IRNSGLEEASLPF (ACPR)
3 (4.9)IRNSGLEEASLCF (ACPR)
3 (4.9)IRNSGLEEASLost
1 (1.6)IRNSGLEKASACPR
1 (1.6)IRNSGLE/KEASACPR
3 (4.9)IRNSGLKEASACPR
6 (9.8)IRNA/FGLEEASACPR
1 (1.6)IRNA/FGLEKASACPR
1 (1.6)IRNA/FGLKEASACPR
1 (1.6)IRNA/FGLKEASLost
1 (1.6)IRNA/FGLE/KEASACPR
4 (6.6)IRNS + A/FGLEEASACPR

dhfr = dihydrofolate reductase; dhps = dihydropteroate synthase; atp6 = ATPase 6; ACPR = adequate clinical and parasitologic response; LPF = late parasitologic failure; LCF = late clinical failure; Lost = lost-to-follow-up. Mutant alleles are indicated in bold. Triple dhfr mutant is defined by the haplotype IRN (Ile-51, Arg-59, Asn-108). Quadruple mutant is defined by the dhfr-dhps haplotype IRN + G (triple dhfr mutant + Gly-437). All isolates carried the wild-type DHFR Ile-164 and DHPS Lys-540 alleles. In the presence of Ala-436 or mixed Ser- and Ala-436 in DHPS, the presence or absence of Phe-436 cannot be established with restriction enzymes Mnl I and MspA1 I.

Recrudescence.

Reinfection (classified as ACPR after polymerase chain reaction correction).

Discussion

The results of the present study showed that all isolates are triple dhfr mutants and wild-type or single dhps mutant at position 437. The role of dhps mutant alleles Ser436Ala or Ser436Phe in African isolates is not well known. The predominance of triple dhfr mutants and the absence of Ile164Leu in DHFR and Lys540Gln in DHPS in isolates from Cameroon are consistent with that of our previous studies on isolates obtained during 1999–2004, in which we observed a steady replacement of wild-type parasites (52% in 1994–1995 and only 3% in 2004–2005) by triple dhfr mutants.14

Studies conducted with large numbers of P. falciparum isolates in eastern and western Africa and Asia (China, Cambodia) have shown E431K, N569K, and A630S substitutions (also additional rare mutations) in the pfatp6 gene but did not find any mutant with L263E, A623E, or S769N, despite the fact that artemisinin derivatives have been used extensively in Asia.1519 Only one African isolate carrying the South American-type S769N substitution has been reported, but its low dihydroartemisinin IC50 (0.83 nM) suggested full in vitro sensitivity.20 In isolates from Cameroon, only E431K, E432K, E641Q, and E643Q changes have been observed.13 The novel E432K mutation, which occurred in the background of a wild-type LEAS haplotype, was also observed in our earlier study of isolates from Cameroon obtained during 2001–2006.13 The presence of these mutations did not influence the level of dihydroartemisinin IC50. The pfatp6 gene seems to show polymorphic patterns depending on the geographic origin of parasites, but these mutations, including changes in amino acid residues 263, 431, 623, and 769, have not been consistently associated with changes in artemisinin IC50 level or poor clinical response to ACT.

The AS-SP combination was shown to be highly effective in Cameroon. Elsewhere in Africa, where SP is less efficacious, AS-SP is not the recommended ACT. This discordance may be explained, at least in part, by the absence of additional dhfr and dhps mutations known to increase the level of antifolate resistance (DHFR-164 and DHPS-540) in P. falciparum isolates from Cameroon and lack of molecular evidence for artemisinin resistance on the basis of pfatp6 analysis. In the present study, recrudescence occurred in patients infected with triple or quadruple dhfr-dhps mutant and wild-type pfatp6 parasite, and patients infected with quadruple dhfr-dhps mutants were cured with AS-SP. These results, and those of other studies, suggest that mutations in genetic markers for drug resistance are necessary but not sufficient cause that leads to treatment failure. Field isolates of P. falciparum remain highly sensitive in vitro to artemisinin derivatives. However, the use of pfatp6 as a molecular marker and conventional in vitro assays may not be appropriate tools to detect artemisinin resistance. If quiescence mechanism is demonstrated in naturally occurring P. falciparum isolates,6 new alternative laboratory tools are required to determine artemisinin-resistant phenotype.

ACKNOWLEDGMENTS:

We thank the personnel of the Nlongkak Catholic Mission Dispensary in Yaoundé, Cameroon, for assistance in recruiting patients.

  • 1.

    World Health Organization, 2005. Susceptibility of Plasmodium falciparum to Antimalarial Drugs. Report on Global Monitoring 1996–2004: Geneva: World Health Organization.

    • Search Google Scholar
    • Export Citation
  • 2.

    Dondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, von Seidlein L, 2010. Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol 8: 272280.

    • Search Google Scholar
    • Export Citation
  • 3.

    Krishna S, Pulcini S, Fatih F, Staines H, 2010. Artemisinins and the biological basis for the PfATP6/SERCA hypothesis. Trends Parasitol 26: 517523.

    • Search Google Scholar
    • Export Citation
  • 4.

    Uhlemann AC, Cameron A, Eckstein-Ludwig U, Fischbarg J, Iserovich P, Zuniga FA, East M, Lee A, Brady L, Haynes RK, Krishna S, 2005. A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nat Struct Mol Biol 12: 628629.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O, 2005. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 366: 19601963.

    • Search Google Scholar
    • Export Citation
  • 6.

    Witkowski B, Lelièvre J, Lopez Barragan MJ, Laurent V, Su XZ, Berry A, Benoit-Vical F, 2010. Increased tolerance to artemisinin in Plasmodium falciparum is mediated by a quiescence mechanism. Antimicrob Agents Chemother 54: 18721877.

    • Search Google Scholar
    • Export Citation
  • 7.

    Pearce RJ, Pota H, Evehe MSB, Ba EH, Mombo-Ngoma G, Malisa AL, Ord R, Inojosa W, Matondo A, Diallo DA, Mbacham W, van den Broek IV, Swarthout TD, Getachew A, Dejene S, Grobusch MP, Njie F, Dunyo S, Kweku M, Owusu-Agyei S, Chandramohan D, Bonnet M, Guthmann JP, Clarke S, Barnes KI, Streat E, Katokele ST, Uusiku P, Agboghoroma CO, Elegba OY, Cisse B, A-Elbasit IE, Giha HA, Kachur SP, Lynch C, Rwakimari JB, Chanda P, Hawela M, Sharp B, Naidoo I, Roper C, 2009. Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria. PLoS Med 6: e1000055.

    • Search Google Scholar
    • Export Citation
  • 8.

    Whegang SY, Tahar R, Foumane VN, Soula G, Gwet H, Thalabard JC, Basco LK, 2010. Efficacy of non-artemisinin- and artemisinin-based combination therapies for uncomplicated malaria in Cameroon. Malar J 9: 56. Available at: http://www.malariajournal.com/content/9/1/56.

    • Search Google Scholar
    • Export Citation
  • 9.

    World Health Organization, 2003. Assessment and Monitoring of Antimalarial Drug Efficacy for the Treatment of Uncomplicated falciparum Malaria. Geneva: World Health Organization, WHO/HTM/RBM/2003.50.

    • Search Google Scholar
    • Export Citation
  • 10.

    World Health Organization, 2008. Methods and Techniques for Clinical Trials on Antimalarial Drug Efficacy: Genotyping to Identify Parasite Populations. Informal Consultation Organized by the Medicines for Malaria Venture and Cosponsored by the World Health Organization, May 29–31, 2007, Amsterdam, The Netherlands.

    • Search Google Scholar
    • Export Citation
  • 11.

    Eldin de Pécoulas P, Basco LK, Abdallah B, Djé MK, Le Bras J, Mazabraud A, 1995. Plasmodium falciparum: detection of antifolate resistance by mutation-specific restriction enzyme digestion. Exp Parasitol 80: 483487.

    • Search Google Scholar
    • Export Citation
  • 12.

    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: 18.

    • Search Google Scholar
    • Export Citation
  • 13.

    Tahar R, Ringwald P, Basco LK, 2009. Molecular epidemiology of malaria in Cameroon. XXVIII. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum and sequence analysis of the P. falciparum ATPase 6 gene. Am J Trop Med Hyg 81: 1318.

    • Search Google Scholar
    • Export Citation
  • 14.

    Tahar R, Basco LK, 2007. Molecular epidemiology of malaria in Cameroon. XXVII. Clinical and parasitological response to sulfadoxine-pyrimethamine treatment and Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase alleles in Cameroonian children. Acta Trop 103: 8189.

    • Search Google Scholar
    • Export Citation
  • 15.

    Mugittu K, Genton B, Mshinda H, Beck HP, 2006. Molecular monitoring of Plasmodium falciparum resistance to artemisinin in Tanzania. Malar J 5: 126. Available at: http://www.malariajournal.com/content/5/1/126.

    • Search Google Scholar
    • Export Citation
  • 16.

    Dahlström S, Veiga MI, Ferreira P, Mårtensson A, Kaneko A, Andersson B, Björkman A, Gil JP, 2008. Diversity of the sarco/endoplasmic reticulum Ca2+-ATPase orthologue of Plasmodium falciparum (PfATP6). Infect Genet Evol 8: 340345.

    • Search Google Scholar
    • Export Citation
  • 17.

    Menegon M, Sannella AR, Majori G, Severini C, 2008. Detection of novel point mutations in the Plasmodium falciparum ATPase 6 candidate gene for resistance to artemisinins. Parasitol Int 57: 233235.

    • Search Google Scholar
    • Export Citation
  • 18.

    Zhang GQ, Guan Y, Zheng B, Wu S, Tang LH, 2008. No PfATPase6 S769N mutation found in Plasmodium falciparum isolates from China. Malar J 7: 122. Available at: http://www.malariajournal.com/content/7/1/112.

    • Search Google Scholar
    • Export Citation
  • 19.

    Ibrahim ML, Khim N, Adam HH, Ariey F, Duchemin JB, 2009. Polymorphism of PfATPase in Niger: detection of three new point mutations. Malar J 8: 28. Available at: http://www.malariajournal.com/content/8/1/28.

    • Search Google Scholar
    • Export Citation
  • 20.

    Cojean S, Hubert V, Le Bras J, Durand R, 2006. Resistance to dihydroartemisinin. Emerg Infect Dis 12: 17981799.

Author Notes

*Address correspondence to Leonardo Basco, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Institut de Recherche pour le Développement, Yaoundé, Cameroon. E-mail: lkbasco@yahoo.fr

Financial support: This study was supported by the French Agence Nationale de la Recherche (project RES-ATQ, ANR-08-MIE-024).

Authors' addresses: Virginie Menemedengue, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Institut de Recherche pour le Développement, Yaoundé, Cameroon and Département de Biologie Animale et Physiologie, Université de Yaoundé I, Yaoundé, Cameroon. Khalifa Sahnouni, Unité Mixte de Recherche 216, Institut de Recherche pour le Développement, Université René Descartes-Paris V, Paris, France. Leonardo Basco, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), Institut de Recherche pour le Développement, Yaoundé, Cameroon and Unité Mixte de Recherche 198, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Institut de Recherche pour le Développement, Université Aix-Marseille 2, Marseille, France. Rachida Tahar, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Institut de Recherche pour le Développement, Yaoundé, Cameroon and Unité Mixte de Recherche 216, Institut de Recherche pour le Développement, Université René Descartes-Paris V, Paris, France.

Save