• 1

    Mendis K, Sina BJ, Marchesini P, Carter R, 2001. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 64 :97–106.

  • 2

    Hay SI, Guerra CA, Tatem AJ, Noor AM, Snow RW, 2004. The global distribution and population at risk of malaria: past, present, and future. Lancet Infect Dis 4 :327–336.

    • Search Google Scholar
    • Export Citation
  • 3

    Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM, 2007. Vivax malaria: neglected and not benign. Am J Trop Med Hyg 77 :79–87.

    • Search Google Scholar
    • Export Citation
  • 4

    Duarte EC, Gyorkos TW, Pang L, Abrahamowicz M, 2004. Epidemiology of malaria in a hypoendemic Brazilian Amazon migrant population: a cohort study. Am J Trop Med Hyg 70 :229–237.

    • Search Google Scholar
    • Export Citation
  • 5

    Legrand E, Volney B, Meynard JB, Mercereau-Puijalon O, Esterre P, 2008. In vitro monitoring of Plasmodium falciparum drug resistance in French Guiana: a synopsis of continuous assessment from 1994 to 2005. Antimicrob Agents Chemother 52 :288–298.

    • Search Google Scholar
    • Export Citation
  • 6

    Carme B, 2005. Substantial increase of malaria in inland areas of eastern French Guiana. Trop Med Int Health 10 :154–159.

  • 7

    Picot S, 2006. Is Plasmodium vivax still a paradigm for uncomplicated malaria? Med Mal Infect 36 :406–413.

  • 8

    D’Ortenzio E, Durand R, Pradines B, Parzy D, Lebras J, 2005. Dernières recommandations sur la prévention et le traitement du paludisme. Available at: http://www.phans.asso.fr/Documents%20partages/Journees%20medicales/PALUDISMEpc.pdf. Accessed January 13, 2009.

  • 9

    Cortese JF, Caraballo A, Contreras CE, Plowe CV, 2002. Origin and dissemination of Plasmodium falciparum drug-resistance mutations in South America. J Infect Dis 186 :999–1006.

    • Search Google Scholar
    • Export Citation
  • 10

    Alecrim WD, Dourado H, Alecrim MG, Passos LF, Wanssa E, Albuquerque B, 1982. In vivo resistance of Plasmodium falciparum to the combination of sulfadoxine and pyrimethamine, at RIII level, in Amazonas, Brazil. Rev Inst Med Trop Sao Paulo 24 :52–53.

    • Search Google Scholar
    • Export Citation
  • 11

    Espinal CA, Cortes GT, Guerra P, Arias AE, 1985. Sensitivity of Plasmodium falciparum to antimalarial drugs in Colombia. Am J Trop Med Hyg 34 :675–680.

    • Search Google Scholar
    • Export Citation
  • 12

    Roper MH, Torres RS, Goicochea CG, Andersen EM, Guarda JS, Calampa C, Hightower AW, Magill AJ, 2000. The epidemiology of malaria in an epidemic area of the Peruvian Amazon. Am J Trop Med Hyg 62 :247–256.

    • Search Google Scholar
    • Export Citation
  • 13

    Prajapati SK, Joshi H, Valecha N, Reetha AM, Eapen A, Kumar A, Das MK, Yadav RS, Rizvi MA, Dash AP, 2007. Allelic polymorphism in the Plasmodium vivax dihydrofolate reductase gene among Indian field isolates. Clin Microbiol Infect 13 :331–334.

    • Search Google Scholar
    • Export Citation
  • 14

    Alam MT, Bora H, Bharti PK, Saifi MA, Das MK, Dev V, Kumar A, Singh N, Dash AP, Das B, Wajihullah, Sharma YD, 2007. Similar trends of pyrimethamine resistance-associated mutations in Plasmodium vivax and P. falciparum.Antimicrob Agents Chemother 51 :857–863.

    • Search Google Scholar
    • Export Citation
  • 15

    Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH, 2007. Antifolates can have a role in the treatment of Plasmodium vivax.Trends Parasitol 23 :213–222.

    • Search Google Scholar
    • Export Citation
  • 16

    Legrand E, Volney B, Lavergne A, Tournegros C, Florent L, Accrombessi D, Guillotte M, Mercereau-Puijalon O, Esterre P, 2005. Molecular analysis of two local falciparum malaria outbreaks on the French Guiana coast confirms the msp1 B-K1/varD genotype association with severe malaria. Malar J 4 :26.

    • Search Google Scholar
    • Export Citation
  • 17

    Snounou G, 1996. Detection and identification of the four malaria parasite species infecting humans by PCR amplification. Methods Mol Biol 50 :263–291.

    • Search Google Scholar
    • Export Citation
  • 18

    Imwong M, Pukrittayakamee S, Renia L, Letourneur F, Charlieu JP, Leartsakulpanich U, Looareesuwan S, White NJ, Snounou G, 2003. Novel point mutations in the dihydrofolate reductase gene of Plasmodium vivax: evidence for sequential selection by drug pressure. Antimicrob Agents Chemother 47 :1514–1521.

    • Search Google Scholar
    • Export Citation
  • 19

    Korsinczky M, Fischer K, Chen N, Baker J, Rieckmann K, Cheng Q, 2004. Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site. Antimicrob Agents Chemother 48 :2214–2222.

    • Search Google Scholar
    • Export Citation
  • 20

    Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41 :95–98.

    • Search Google Scholar
    • Export Citation
  • 21

    Targett GAT, 1992. Malaria—drug-use and the immune-response. Parasitology 105 :S61–S70.

  • 22

    Tjitra E, Baker J, Suprianto S, Cheng Q, Anstey NM, 2002. Therapeutic efficacies of artesunate-sulfadoxine-pyrimethamine and chloroquine-sulfadoxine-pyrimethamine in vivax malaria pilot studies: relationship to Plasmodium vivax dhfr mutations. Antimicrob Agents Chemother 46 :3947–5393.

    • Search Google Scholar
    • Export Citation
  • 23

    Hawkins VN, Auliff A, Prajapati SK, Rungsihirunrat K, Hapuarachchi HC, Maestre A, O’Neil MT, Cheng Q, Joshi H, Na-Bangchang K, Sibley CH, 2008. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase. Malar J 7 :72.

    • Search Google Scholar
    • Export Citation
  • 24

    McCollum AM, Mueller K, Villegas L, Udhayakumar V, Escalante AA, 2007. Common origin and fixation of Plasmodium falciparum dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance in a low-transmission area in South America. Antimicrob Agents Chemother 51 :2085–2091.

    • Search Google Scholar
    • Export Citation
  • 25

    Khalil I, Ronn AM, Alifrangis M, Gabar HA, Satti GM, Bygbjerg IC, 2003. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole. Am J Trop Med Hyg 68 :586–589.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 

 

 

 

 

High Prevalence and Fixation of Plasmodium vivax dhfr/dhps Mutations Related to Sulfadoxine/Pyrimethamine Resistance in French Guiana

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  • 1 University Lyon 1, Malaria Research Unit, Lyon, France; National Reference Centre of Malaria Chemoresistance in French Guiana and West Indies (CNRCP), Institut Pasteur de la Guyane, Cayenne, French Guiana; Institut Pasteur, Plate-forme Génomique, Paris, France; Institut de Médecine Tropicale du Service de Santé des Armées, Unité de Recherche en Biologie et Epidémiologie Parasitaires, UMR 6236-URMITE-IMTSSA, Marseille, France; Institut Pasteur de Madagascar, Malaria Research Unit, Antananarivo, Madagascar

Plasmodium vivax isolates from French Guiana were studied for the presence of mutations associated with sulfadoxine/pyrimethamine (SP) drug resistance. Ninety-six blood samples were collected from 2000 to 2005 from symptomatic malaria patients. SP drug resistance was predicted by determining point mutations in the dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) genes. All samples showed mutant genotypes in both genes with a prevalence > 90% for the 58R, 117N, 382C, and 383G. A new mutation (116G) in pvdhfr was found at a frequency of 3.3%. Six different pvdhfr/dhps multilocus genotypes were observed with the predominance of the quintuple mutant-type 58R/117N/173L–382C/383G (59.3%). No significant differences were observed between the prevalence of haplotypes and the year of collection. Our results indicate that, in this area, the fixation of SP drug-resistant parasites in the P. vivax population is stable.

Plasmodium vivax remains the most geographically widespread of the five Plasmodium species infecting humans and the second most common cause of malaria in the world, throughout Asia, South and Central America, the Middle East, and some parts of Africa. It is responsible for 25–40% of the global malaria burden, which may represent between 80 and 400 million cases annually.13 In South America, P. vivax remains the predominant pathogen causing human malaria,4 except in the Guyana Shield, where P. falciparum infection is more frequent.5 In French Guiana, P. vivax morbidity accounts for > 50% of the 3,000–3,500 malaria cases reported each year until 2007, particularly among ethnic groups who carry the Duffy antigen such as Asians or Amerindians. 6,7 Since 2002, the artemether-lumefantrine combination is recommended as first-line treatment in uncomplicated falciparum malaria, whereas chloroquine is used to treat vivax malaria, despite the absence of data on antimalarial drug efficacy.8

Chloroquine and pyrimethamine were used widely in South America during the 1950s and the 1960s, either as treatment or prophylactics against malaria.9 They were withdrawn and replaced in the 1970s by the sulfadoxine/pyrimethamine (SP) drug combination to treat malaria cases caused by chloroquine-resistant P. falciparum isolates. This antifolates combination acts synergistically to inhibit the folate biosynthesis pathway in both P. falciparum and P. vivax, as competitive inhibitors of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). However, since then, P. falciparum resistance to SP has been reported in many locations in South America including Colombia, Brazil, and Peru. 1012 In other parts of the world, the intensive use of antifolates such as SP against P. falciparum malaria has selected P. vivax dhfr and dhps mutant parasites 13,14 and raises potential that mutations related to SP resistance will increase in prevalence among South America P. vivax isolates. In this region, data are lacking mainly because large-scale surveys for SP resistance mutations in P. vivax isolates have not been performed. Indeed, to date, only 19 isolates, collected between 1972 and 2005, have been assessed (RFLP or DNA sequencing) for dihydrofolate reductase (pvdhfr) polymorphisms.15

To improve surveillance and track the spread of the SP resistance in P. vivax malaria in this part of the world, the objective of this study was to determine the prevalence of pvdhfr and pvdhps mutations of P. vivax isolates collected between 2000 and 2005 from various endemic areas of French Guiana.

All studied isolates were sent to the malaria reference center in Pasteur Institute of French Guiana in Cayenne (CNRCP) by private practitioners, health centers, and/or hospitals, as recommended by health authorities to follow drug resistance in Plasmodium spp. A 2- to 5-mL blood sample was collected from each patient by venipuncture in K +EDTA vacutainers before treatment. Blood samples were maintained at +4°C during shipment to the Pasteur Institute, where they were immediately processed (Giemsa-stained thin/thick blood films, DNA extracted 16) to confirm P. vivax infection by microscopy and polymerase chain reaction (PCR) as previously described by Snounou. 17 Ninety-six isolates, originating from peripheral health centers located along the Oyapock river (Brazil boarder) and from the main hospitals on the coast (mainly in Cayenne), were identified as monospecific P. vivax infections. P. vivax isolates were genotyped for the pvdhfr and pvdhps genes. DNA was amplified by semi-nested PCR for pvdhfr or by nested PCR for pvdhps using gene-specific primers. 18,19 PCR products were purified, using polyacrylamide P-100 Gel (Bio-Gel P-100; BioRad, Marnes-la-Coquette, France) by 96-well plate filtration (Millipore, St. Quentin en Yvelines, France). Sequencing reactions were performed using ABI PRISM BigDye Terminator cycle sequencing ready reactions kit and run on a 3730 xl Genetic Analyzer (Applied Biosystems, Courtaboeuf, France). Electrophoregrams were visualized and analyzed with CEQ2000 Genetic Analysis System software (Beckman Coulter, Villepinte, France). Nucleotide sequences for field isolates were compared with wild-type sequences (GenBank accession no. X98123 for pvdhfr and AY186730 for pvdhps), using BioEdit Sequence Alignment Editor software. 20 Isolates with mixed alleles (both wild-type and mutant alleles present) were considered as mutant in the finale interpretation. A second PCR product was sequenced for confirmation if a new point mutation was observed. The complete sequences of the alleles identified have been submitted to GenBank and assigned accession numbers FJ384768–FJ384777 for pvdhfr and FJ389049–FJ389050 for pvdhps.

Among the 96 samples analyzed, 90 were successfully amplified and sequenced for pvdhfr and pvdhps genes. For the pvdhfr gene, three synonymous mutations were identified at codon 15 (6.7%, GCA → GCG, including 2.2% mixed alleles), codon 19 (3.3%, GTC → GTT), and codon 69 (85.6%, TAT → TAC). Four non-synonymous mutations were observed: three previously described at codons 58 (98.9%, S → R encoded either by AGA, AGG, or CGT, including 2.2% mutant mixed alleles), 117 (100%, S → N) and 173 (34.4%, I → L, including 3.3% mixed alleles), and one new at codon 116 (3.3%, S → G). Compared with the wild-type pvdhfr allele 57F/58S/116S/117S/173I, four mutant alleles were found (amino-acids conferring resistance are underlined): one single-mutant genotype 57F/58S/116S/117N/173I (1.1%), one double-mutant genotype 57F/58R/116S/117N/173I (34.9%), and two triple-mutant genotypes 57F/58R/116S/117N/173L (60.5%) and 57F/58R/116G/117N/173I (3.5%). No significant differences were observed in prevalence of pvdhfr mutant genotypes among the years of P. vivax sampling (Fisher exact test, P = 0.48). Taking into consideration both synonymous and non-synonymous mutations, 10 different genotypes were identified.

For pvdhps, two non-synonymous mutations were identified at codons 382 (94.4%, S → C) and 383 (100%, A → G). Only one isolate with 382S/C and 383A/G mixed alleles was observed. Five isolates were found to carry only the 383G mutation, whereas 81 isolates (90.0%) carried a double-mutant 382C/383G allele. These 382C/383G double mutant-type isolates carried the same tandem repeat sequence as the reference sequence (tandem repeat region located between amino acid residues 603 and 666). The five single-mutant isolates showed a 42-nucleotide deletion. Prevalences of the single nucleotide polymorphisms (SNPs) and multilocus genotypes in pvdhfr and pvdhps genes are detailed in Table 1.

Interestingly, in this initial study of P. vivax dhfr and dhps polymorphisms, we found evidence of no wild-type alleles in our isolates for the pvdhfr gene and potentially only one for pvdhps, but in this case, it was present as mixed infection, and the genotypes remain undetermined. Despite the limited record of SP use in this area (accurate data on SP use in our study population or in neighboring countries are not available), the quintuple mutant-type (pvdhfr 58R/117N/173L, pvdhps 382C/383G) involved in SP resistance15,19 was largely predominant. Such prevalence has been was already observed in several regions of the world including South America (Brazil, Columbia), Southeast Asia (Cambodia, China, Myanmar, Philippines, Thailand, Vietnam), India, or South Pacific (Indonesia, Papua New Guinea). 15 Even, it is impossible to draw conclusions about the probable efficacy of SP, in vitro association studies between pvdhfr alleles and pyrimethamine resistance have shown that the 58R/117N and the 58R/117N/173L mutant enzymes were, respectively, ~100- to 460-fold and ~500-fold more pyrimethamine resistant than wild types. 15 However, as has been observed in P. falciparum malaria, the correlation between pvdhfr/dhps multilocus genotypes and the clinical response to SP treatment is markedly discordant, particularly because of the influence of host factors. 21

Besides the mutations previously described, we identified a new mutation at codon 116 in the pvdhfr gene in three isolates collected in 2000/2001 leading to the replacement of serine (polar amino acid) by glycine (non-polar amino acid). To assess the role of this mutation in SP drug resistance, the mutant DHFR should be expressed using a yeast heterologous system. 15

Among the 10 pvdhfr genotypes observed in our study, the 173L and 116G amino acid substitutions were not found in the same genotype. However, it remains difficult to suggest different stepwise processes occurring, as has been already described in Papua 22 because of the too low number of 116G mutant isolates. The double mutant 58R/117N genotype observed in French Guiana was found in six different genotypes and the triple mutant 58R/117N/173L in two different genotypes, leading to the conclusion that triple mutations have arisen at least two times. That is consistent with recent observations of Hawkins and others. 23 As has been observed in P. falciparum malaria in South America, 24 we found pvdhfr mutations (58R and 117N) and pvdhps mutations (382C and 383G) are approaching fixation in French Guiana P. vivax parasite populations, even though the only antifolate used in this country in the past or currently is proguanil in combination therapies (chloroquine/proguanil or atovaquone/proguanil). However, as has been mentioned by McCollum and others 24 in Venezuela, the use of cotrimoxazole (CTX; trimethoprim/sulfamethoxazole) as a drug to treat respiratory infections or diarrhea may be responsible for maintaining selective pressure for the high prevalence of SP-resistant alleles, resulting from cross-resistance between trimethoprim and pyrimethamine, sulfamethoxazole, and sulfadoxine. 25

In summary, our results indicate that, in a low-transmission area, the fixation of SP drug-resistant parasites in the P. vivax population is stable, even in the absence of SP drug pressure, according to the current guidelines. These findings highlight the importance of careful management of anti-malarial drug use to avoid situations that might render the available drugs permanently ineffective as a mean of malaria control. Further studies on allelic diversity at flanking microsatellite loci of P. vivax dhfr and dhps genes will be helpful in understanding the origin and spread of antifolate resistance in this area.

Table 1

Prevalences of the SNPs and multilocus genotypes in P. vivax dhfr and dhps genes in isolates collected from 2000 to 2005 in French Guiana, South America

Table 1

*

Address correspondence to Didier Ménard, Malaria Research Unit, Institut Pasteur de Madagascar, BP 1274, Antananarivo 101, Madagascar. E-mail: dmenard@pasteur.mg

Authors’ addresses: Céline Barnadas and Stéphane Picot, University Lyon 1, EA4170, Malaria Research Unit, Lyon, France. Lise Musset and Eric Legrand, National Reference Centre of Malaria Chemoresistance in French Guiana and West Indies (CNRCP), Institut Pasteur de la Guyane, Cayenne, French Guiana. Magali Tichit and Christiane Bouchier, Institut Pasteur, Plate-forme Génomique, Paris, France. Sébastien Briolant, Thierry Fusai, and Christophe Rogier, Institut de Médecine Tropicale du Service de Santé des Armées, Unité de Recherche en Biologie et Epidémiologie Parasitaires, UMR 6236-URMITE,-IMTSSA, Marseille, France. Didier Ménard, Malaria Research Unit, Institut Pasteur de Madagascar, BP 1274, Antanarivo 101, Madagascar, Tel: 261-20-22-412-72, Fax: 261-20-22-415-34, E-mail: dmenard@pasteur.mg.

Acknowledgments: The authors thank the patients and healthcare workers involved in the network for the surveillance of malaria resistance in French Guiana from which these samples were obtained. The authors thank Peter A. Zimmerman for critical comments and review of this manuscript.

Financial support: This study was supported by grants from French Army DGA 06co006, Natixis/Impact Malaria through the Observatoire de la Résistance aux Antipaludiques Project (ORA), and the Genomics Platform, Pasteur Génopôle, Institut Pasteur, Paris, France. Céline Barnadas is a PhD student supported by the Fondation Jeunesse Internationale (Fondation de France), BioMérieux “Prix BioMérieux infectiologie 2006,” Association des Internes et Anciens Internes en Pharmacie des Hôpitaux de Lyon “Prix R. Rizard,” and the Hospices Civils de Lyon.

REFERENCES

  • 1

    Mendis K, Sina BJ, Marchesini P, Carter R, 2001. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 64 :97–106.

  • 2

    Hay SI, Guerra CA, Tatem AJ, Noor AM, Snow RW, 2004. The global distribution and population at risk of malaria: past, present, and future. Lancet Infect Dis 4 :327–336.

    • Search Google Scholar
    • Export Citation
  • 3

    Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM, 2007. Vivax malaria: neglected and not benign. Am J Trop Med Hyg 77 :79–87.

    • Search Google Scholar
    • Export Citation
  • 4

    Duarte EC, Gyorkos TW, Pang L, Abrahamowicz M, 2004. Epidemiology of malaria in a hypoendemic Brazilian Amazon migrant population: a cohort study. Am J Trop Med Hyg 70 :229–237.

    • Search Google Scholar
    • Export Citation
  • 5

    Legrand E, Volney B, Meynard JB, Mercereau-Puijalon O, Esterre P, 2008. In vitro monitoring of Plasmodium falciparum drug resistance in French Guiana: a synopsis of continuous assessment from 1994 to 2005. Antimicrob Agents Chemother 52 :288–298.

    • Search Google Scholar
    • Export Citation
  • 6

    Carme B, 2005. Substantial increase of malaria in inland areas of eastern French Guiana. Trop Med Int Health 10 :154–159.

  • 7

    Picot S, 2006. Is Plasmodium vivax still a paradigm for uncomplicated malaria? Med Mal Infect 36 :406–413.

  • 8

    D’Ortenzio E, Durand R, Pradines B, Parzy D, Lebras J, 2005. Dernières recommandations sur la prévention et le traitement du paludisme. Available at: http://www.phans.asso.fr/Documents%20partages/Journees%20medicales/PALUDISMEpc.pdf. Accessed January 13, 2009.

  • 9

    Cortese JF, Caraballo A, Contreras CE, Plowe CV, 2002. Origin and dissemination of Plasmodium falciparum drug-resistance mutations in South America. J Infect Dis 186 :999–1006.

    • Search Google Scholar
    • Export Citation
  • 10

    Alecrim WD, Dourado H, Alecrim MG, Passos LF, Wanssa E, Albuquerque B, 1982. In vivo resistance of Plasmodium falciparum to the combination of sulfadoxine and pyrimethamine, at RIII level, in Amazonas, Brazil. Rev Inst Med Trop Sao Paulo 24 :52–53.

    • Search Google Scholar
    • Export Citation
  • 11

    Espinal CA, Cortes GT, Guerra P, Arias AE, 1985. Sensitivity of Plasmodium falciparum to antimalarial drugs in Colombia. Am J Trop Med Hyg 34 :675–680.

    • Search Google Scholar
    • Export Citation
  • 12

    Roper MH, Torres RS, Goicochea CG, Andersen EM, Guarda JS, Calampa C, Hightower AW, Magill AJ, 2000. The epidemiology of malaria in an epidemic area of the Peruvian Amazon. Am J Trop Med Hyg 62 :247–256.

    • Search Google Scholar
    • Export Citation
  • 13

    Prajapati SK, Joshi H, Valecha N, Reetha AM, Eapen A, Kumar A, Das MK, Yadav RS, Rizvi MA, Dash AP, 2007. Allelic polymorphism in the Plasmodium vivax dihydrofolate reductase gene among Indian field isolates. Clin Microbiol Infect 13 :331–334.

    • Search Google Scholar
    • Export Citation
  • 14

    Alam MT, Bora H, Bharti PK, Saifi MA, Das MK, Dev V, Kumar A, Singh N, Dash AP, Das B, Wajihullah, Sharma YD, 2007. Similar trends of pyrimethamine resistance-associated mutations in Plasmodium vivax and P. falciparum.Antimicrob Agents Chemother 51 :857–863.

    • Search Google Scholar
    • Export Citation
  • 15

    Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH, 2007. Antifolates can have a role in the treatment of Plasmodium vivax.Trends Parasitol 23 :213–222.

    • Search Google Scholar
    • Export Citation
  • 16

    Legrand E, Volney B, Lavergne A, Tournegros C, Florent L, Accrombessi D, Guillotte M, Mercereau-Puijalon O, Esterre P, 2005. Molecular analysis of two local falciparum malaria outbreaks on the French Guiana coast confirms the msp1 B-K1/varD genotype association with severe malaria. Malar J 4 :26.

    • Search Google Scholar
    • Export Citation
  • 17

    Snounou G, 1996. Detection and identification of the four malaria parasite species infecting humans by PCR amplification. Methods Mol Biol 50 :263–291.

    • Search Google Scholar
    • Export Citation
  • 18

    Imwong M, Pukrittayakamee S, Renia L, Letourneur F, Charlieu JP, Leartsakulpanich U, Looareesuwan S, White NJ, Snounou G, 2003. Novel point mutations in the dihydrofolate reductase gene of Plasmodium vivax: evidence for sequential selection by drug pressure. Antimicrob Agents Chemother 47 :1514–1521.

    • Search Google Scholar
    • Export Citation
  • 19

    Korsinczky M, Fischer K, Chen N, Baker J, Rieckmann K, Cheng Q, 2004. Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site. Antimicrob Agents Chemother 48 :2214–2222.

    • Search Google Scholar
    • Export Citation
  • 20

    Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41 :95–98.

    • Search Google Scholar
    • Export Citation
  • 21

    Targett GAT, 1992. Malaria—drug-use and the immune-response. Parasitology 105 :S61–S70.

  • 22

    Tjitra E, Baker J, Suprianto S, Cheng Q, Anstey NM, 2002. Therapeutic efficacies of artesunate-sulfadoxine-pyrimethamine and chloroquine-sulfadoxine-pyrimethamine in vivax malaria pilot studies: relationship to Plasmodium vivax dhfr mutations. Antimicrob Agents Chemother 46 :3947–5393.

    • Search Google Scholar
    • Export Citation
  • 23

    Hawkins VN, Auliff A, Prajapati SK, Rungsihirunrat K, Hapuarachchi HC, Maestre A, O’Neil MT, Cheng Q, Joshi H, Na-Bangchang K, Sibley CH, 2008. Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase. Malar J 7 :72.

    • Search Google Scholar
    • Export Citation
  • 24

    McCollum AM, Mueller K, Villegas L, Udhayakumar V, Escalante AA, 2007. Common origin and fixation of Plasmodium falciparum dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance in a low-transmission area in South America. Antimicrob Agents Chemother 51 :2085–2091.

    • Search Google Scholar
    • Export Citation
  • 25

    Khalil I, Ronn AM, Alifrangis M, Gabar HA, Satti GM, Bygbjerg IC, 2003. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole. Am J Trop Med Hyg 68 :586–589.

    • Search Google Scholar
    • Export Citation
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