• 1

    Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LMB, Singh Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE, 2000. Mutations n 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
  • 2

    Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344 :257–263.

    • Search Google Scholar
    • Export Citation
  • 3

    Mehlotra RK, Fujioka H, Roepe PD, Janeeh O, Ursos LMB, Jacobs-Lorena V, McNamara DT, Bockarie MJ, Kazura JW, Kyle DE, Fidock DA, Zimmerman PA, 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci USA 98 :12689–12694.

    • Search Google Scholar
    • Export Citation
  • 4

    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
  • 5

    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
  • 6

    Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewsky C, Kawamoto F, Miller RS, Meshnick. 2003. Resistance to antimalarials in southeast Asia and genetic polymorphisms in pfmdr1.Antimicrobial Agents Chemother 47 :2418–2423.

    • Search Google Scholar
    • Export Citation
  • 7

    World Health Organization, 1990. The Clinical Management of Acute Malaria. Third edition. New Dehli: World Health Organization. Regional Publications, South East Asia Series No. 9.

  • 8

    Sumawinata IW, Bernadeta, Leksana B, Sutamihardja A, Purnomo, Subianto B, Sekartuti, Fryauff DJ, Baird JK. 2003. Very high risk of therapeutic failure with chloroquine for uncomplicated Plasmodium falciparum and Plasmodium vivax malaria in Indonesian Papua. Am J Trop Med Hyg 68 :416–420.

    • Search Google Scholar
    • Export Citation
  • 9

    Huaman MC, Roncal N, Nakazawa S, Long TTA, Gerena L, Garcia C, Solari L, Magill AJ, Kanbara H, 2004. Polymorphism of the Plasmodium falciparum multidrug resistance 1 and chloroquine resistance transporter genes and in vitro susceptibility to aminoquinolines in isolates from the Peruvian Amazon. Am J Trop Med Hyg 70 :1–6.

    • Search Google Scholar
    • Export Citation
  • 10

    Viriyakosol S, Siripoon N, Petcharapirat C, Petcharapirat P, Jarra W, Thaithong S, Brown KN, Snounou G, 1995. Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies. Bull World Health Organ 73 :85–95.

    • Search Google Scholar
    • Export Citation
  • 11

    Gomez-Saladin E, Fryauff DJ, Taylor WR, Laksana BS, Susanti AI, Purnomo, Subianto B, Richie TL. 1999. Plasmodium falciparum mdr1 mutations and in vivo chloroquine resistance in Indonesia. Am J Trop Med Hyg 61 :240–244.

    • Search Google Scholar
    • Export Citation
  • 12

    Nagesha HS, Din-Syafruddin, Casey GJ, Susanti AI, Fryauff DJ. 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 :43–49.

    • Search Google Scholar
    • Export Citation
  • 13

    Maguire DJ, Susanti AI, Krisin, Sismadi P, Fryauff DJ, Baird JK. 2001. The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia. Ann Trop Med Parasitol 95 :559–572.

    • Search Google Scholar
    • Export Citation
  • 14

    Plowe C, 2002. University of Maryland, Baltimore, MD. Web site: http://medschool.umaryland.edu/cvd/2002_pcr_asra.htm.

  • 15

    Cooper RA, Ferdig MT, Su XZ, Ursos LMB, Mu J, Nomura T, Fujioka H, Fidock DA, Roepe PD, Wellems TE, 2002. Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum.Mol Pharmacol 61 :35–42.

    • Search Google Scholar
    • Export Citation
  • 16

    Chen N, Russell B, Staley J, Kotecka B, Nasveld P, Cheng Q, 2001. Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum.J Infect Dis 183 :1543–1545.

    • Search Google Scholar
    • Export Citation
  • 17

    Chen N, Baker J, Ezard N, Burns M, Edstein MD, Cheng Q, 2002. Molecular evaluation of the efficacy of chloroquine treatment of uncomplicated Plasmodium falciparum malaria in East Timor. Am J Trop Med Hyg 67 :64–66.

    • Search Google Scholar
    • Export Citation
  • 18

    Lim P, Chy S, Ariey F, Incardona S, Chim P, Sem R, Denis MB, Hewitt S, Hoyer S, Socheat D, Merecreau-Puijalon O, Fandeur T, 2003. Pfcrt polymorphism and chloroquine resistance in Plasmodium falciparum strains isolated in Cambodia. Antimicrobial Agents Chemother 47 :87–94.

    • Search Google Scholar
    • Export Citation

 

 

 

 

SHORT REPORT: POLYMORPHISMS IN THE CHLOROQUINE RESISTANCE TRANSPORTER GENE IN PLASMODIUM FALCIPARUM ISOLATES FROM LOMBOK, INDONESIA

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  • 1 Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki, Japan; Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru; Meninting Health Center, West Lombok, Indonesia

The polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) and P. falciparum chloroquine resistance transporter (pfcrt) genes, which are associated with chloroquine resistance, were examined in 48 P. falciparum isolates from uncomplicated malaria patients from the West Lombok District in Indonesia. The point mutation N86Y in pfmdr1 was present in 35.4% of the isolates and mutation K76T in pfcrt was found in all but one of the samples studied. Identified pfcrt haplotypes were mainly identical to the Papua New Guinea type SagtVMNT (42 of 48, 87.5%), and a few isolates had the Southeast Asia type CVIET (5 of 48, 10.4%). Moreover, one P. falciparum isolate harbored the K76N mutation, giving rise to the haplotype CVMNN, which was not previously reported in field isolates. Our findings suggest that chloroquine resistance in this area might have the same origin as in Papua New Guinea.

The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum has been investigated and mutations in the P. falciparum CQ resistance transporter gene (pfcrt) located on chromosome 7, and the P. falciparum multidrug resistant gene 1 (pfmdr1), located on chromosome 5, have been implicated. The substitution of threonine for lysine in codon 76, K76T in the pfcrt gene, was shown in vitro to be associated with CQ resistance in isolates from Asia, Africa, South America, and Papua New Guinea.1,2 Sequence polymorphisms at position 72–76 of this gene have been associated with the geographic origin of parasite samples, with the CVIET pattern in resistant isolates from Asia and Africa, and with SVMNT in resistant isolates from Papua New Guinea and South America.1,3 The multidrug resistant gene pfmdr1 with a mutation of asparagine to tyrosine at position 86 (N86Y) has been associated with in vitro resistant strains.4 Although its participation is not clear, it has been suggested that the pfmdr1 mutation may confer some advantage to the parasite in the presence of CQ, thus increasing the level of CQ resistance.2,5 Furthermore, a recent study that included samples from four countries of Southeast Asia described the mutations N86Y in pfmdr1 and K76T in pfcrt genes as molecular markers for predicting clinical outcome of CQ treatment.6

In Indonesia, the first cases of resistance were reported in the early 1970s from Kalimantan and Irian Jaya. Although resistance has been reported on several islands in Indonesia,7 with resistance as high as 95% for P. falciparum and 84% for P. vivax,8 CQ continues to be the first-line treatment of P. falciparum and P. vivax malaria because of its safety and availability at very low cost. Here we examined the prevalence of polymorphisms in the pfmdr1 and pfcrt genes in 48 P. falciparum isolates from the West Lombok District of Indonesia. In addition, the possible origin of CQ resistance in Indonesia is discussed.

Blood samples were collected from 48 patients with uncomplicated P. falciparum malaria in sub-district Batulayar in West Lombok in the West Nusa Tenggara Province of Indonesia (Figure 1) from June to September 2002. Sub-district Batulayar has a population of approximately 35,658. The climate is tropical and malaria transmission occurs more frequently during dry season between April and October, although low-level transmission occurs throughout the year, especially in the hilly-forested ranges of Sidemen to Pusuk. Cases of P. falciparum and P. vivax malaria and a few cases of P. malariae malaria have been reported in the area. The recommended first-line treatment is CQ, 25 mg/kg given over a three-day period. When a treatment failure occurs, the combination of sulfadoxine/pyrimethamine is prescribed. Inclusion criteria were a fever ≥37.5°C during the last 48 hours and a positive result in the NOW ICT® (Binax, Portland, ME) rapid malaria test. Blood samples were collected on filter paper and transported to the Muninting district health center laboratory for malaria testing. Samples positive for P. falciparum malaria were processed thereafter in Japan at the Department of Protozoology of Nagasaki University. Informed consent was obtained from each individual. The study was reviewed and approved by the ethical committee of the Institute of Tropical Medicine of Nagasaki University and the executive committee of the Malaria Control Project in Lombok and Sumbawa under the Japanese International Cooperation Agency partnership program.

The DNA was extracted from filter paper by cutting the blood spot into pieces and soaking them in 0.5% saponin in HBS buffer (140 mM NaCl, 10 mM KCl, 10 mM HEPES, pH 7.2). Thereafter, the QIAamp DNA Kit (Qiagen, Valencia, CA) was used according to the manufacturer’s instructions. The parasite lines FCR3 and K1 were used as controls for the detection of polymorphism at position 86 in pfmdr1 and direct sequencing analysis of pfcrt gene. For genotyping of the glutamate-rich protein (glurp) gene, strains K1 and 3D7 were used as controls in the amplification.

To determine the presence of tyrosine at position 86 in pfmdr1, a nested polymerase chain reaction (PCR)-restriction fragment length polymorphism protocol was used as previously described.9 Digestion with the restriction endonuclease Apo I (New England Biolabs, Inc., Beverly, MA) detects tyrosine at position 86 after resolution of the products by electrophoresis on 1–3% agarose gels (Nusieve 3:1; BioWhittaker Molecular Applications, Rockand, ME). For pfcrt gene analysis, a first amplification was carried out with previously designed primers,9 and the products obtained were used as a template in a nested PCR encompassing the polymorphic codons 72–76 and 97 in exon 2 as previously reported.3 The PCR amplification products were purified using the QIAquick PCR purification kit (Qiagen) and directly sequenced on an ABI310 automated sequencer using ABI PRISM Big Dye Terminator Cycle kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions.

The glurp gene, located on chromosome 11, which has a high degree of polymorphism, was assessed for evaluation of diversity of the P. falciparum isolates population in the region.10 The amplification products were resolved by electrophoresis on a 1% agarose gel and stained with ethidium bromide. The glurp amplification product sizes were estimated using DNAfrag version 3.03 Software (John Nash, Institute for Biologic Sciences, National Research Council of Canada, Ottawa, Ontario, Canada). For comparisons, Fisher’s exact test was used.

Seventeen isolates (35.4%) had the 86Y mutation in the pfmdr1 gene, 26 had wild type N86, and 5 carried both alleles (Table 1). In previous studies in Irian Jaya and West Papua, N86Y was found to show a correlation with CQ-resistant P. falciparum parasites.11,12 The mutation K76T in the pfcrt gene was found in all but one (47 of 48, 97.9%) of the isolates studied. A previous report showed that the K76T mutation showed a correlation with clinical resistance to CQ and as a molecular marker had a sensitivity of 93% and a specificity of 82%.13 Both point mutations in the pfmdr1 and pfcrt genes have been proposed to indicate a tendency toward reduced susceptibility to CQ. Thus, our results suggest potential CQ resistance in the region, although other factors may influence the final treatment outcome. The combination of geographic remoteness to health facilities and lack of interest in seeking medical attention driven by both financial reasons and lack of knowledge resulted in an overall follow-up rate of 23% (11 of 48). After 14 days of CQ treatment, 5 of 11 patients were not able to clear the parasites, and had tendency to harbor 86Y in the pfmdr1 gene (Table 1). However, a larger sample size is required to obtain conclusive results.

A new mutation, K76N, which substitutes asparagine for lysine, was found in one isolate from the sub-village Pusuk, generating the haplotype CVMNN. For confirmation of this finding, independent PCR amplifications and at least three repetitions of sequencing were carried out. In all cases, unambiguous electropherograms were obtained, showing AAT that codes for asparagine at position 76. The K76N mutation could be misidentified as a K76T substitution by a PCR-restriction enzyme protocol.14 To our knowledge, this is the first time that K76N has been reported in a field study. However, it has been reported in laboratory experiments after exposure of parasites to lethal concentrations of CQ. In those experiments, Cooper and others demonstrated that the K76N mutation confers the verapamil-reversible CQ-resistance phenotype associated with greatly reduced accumulation of the drug.15 Contrary to those in vitro experiments, the patient possessing this rare pfcrt haplotype cleared parasites after treatment with CQ. Since other factors participate in the clinical outcome, it would be interesting to look for more isolates with CVMNN and carry out the in vitro susceptibility test.

The sequence analysis of codons 72–76 in the pfcrt gene (Table 1) allowed identification of previously reported haplotypes SagtVMNT (42 of 48, 87.5%) and CVIET (5 of 48, 10.4%). The pfcrt SVMNT haplotype with serine coded by AGT has been found in Bougainville, Papua New Guinea,16 the main island of Papua New Guinea,3 and East Timor.17 The haplotype CVIET has been reported in countries of Southeast Asia.1,18 Since Lombok, Indonesia is located near Papua New Guinea (Figure 1), it is not unexpected that both the SVMNT and CVIET haplotypes were detected.

Furthermore, in our attempt to evaluate the diversity among the isolates studied, we assessed the glurp gene and 11 glurp genotypes were found in West Lombok, ranging from 450 to 1,100 basepairs. The West Lombok District, despite its small area, shows a high degree of diversity in the P. falciparum population that might be a product of high rate of transmission of malaria or human transmigration.

Upon examination for any linkage among the alleles studied in the pfmdr1, pfcrt, and glurp genes, significant associations were found between the pfcrt CVIET haplotype and pfmdr1 86Y (P = 0.0193), the CVIET haplotype and glurp 450 (P < 0.001), and pfmdr1 86Y and glurp 450 (P = 0.0057). Our findings showed that the majority of isolates have pfcrt haplotype SVMNT, pfmdr1 86N, and glurp with molecular masses greater than 450 homogeneously distributed in all the villages from West Lombok, indicating that these might be indigenous in the area. A few isolates harboring pfcrt haplotype CVIET, pfmdr1 86Y, and glurp 450, found mainly in Kedondong Atas village, were most likely introduced recently. Therefore, CQ resistance in Lombok might have the same origin as the Papua New Guinea strains, and the Southeast Asian pfcrt haplotype CVIET might have been introduced only recently in a particular region. Further studies are being carried out in isolates from Lombok and other Indonesian islands to determine the prevalence of the novel K76N mutation and its association with clinical outcome/in vitro susceptibility to CQ.

Table 1

Genotypes for Pfcrt, Pfmdr1, and Glurp genes in 48 Plasmodium falciparum isolates from Lombok, Indonesia*

SubvillagePfcrt haplotype†Codon 86 in Pfmdr1Glurp genotype‡Parasite clearance at 14 days
* Pfcrt = P. falciparum chloroquine resistance transporter; Pfmdr1 = P. falciparum multidrug resistance 1; Glurp = glutamine-rich protein; ND = no data.
† Codons 72–76 in Pfcrt gene.
‡ Polymerase chain reaction product sizes in basepairs.
BuneanSVMNTTyr and Asn650Failure
BuneanSVMNTTyr750Failure
Batu PenyuSVMNTTyr750Failure
Batu Penyu AtasSVMNTAsn750Failure
PelolatSVMNTTyr1000Failure
Kedondong AtasSVMNTAsn700Success
Batu PenyuSVMNTTyr750Success
BatulayarSVMNTAsn700Success
PusukCVMNNAsn950Success
SenggigiSVMNTAsn700Success
SenggigiSVMNTAsn700 and 850Success
PenanggakSVMNTAsn600ND
PenanggakSVMNTAsn800ND
PenanggakSVMNTTyr900ND
PenanggakSVMNTAsn1100ND
PenanggakSVMNTAsn1000ND
PenanggakSVMNTTyr900ND
KekeranSVMNTTyr450ND
KekeranSVMNTTyr750ND
KekeranSVMNTAsn900ND
KekeranSVMNTAsn700ND
Sidemen DayeCVIETTyr450ND
Sidemen DayeSVMNTAsn900ND
Sidemen LaukSVMNTAsn750ND
Sidemen LaukSVMNTAsn550ND
Sidemen LaukSVMNTTyr550ND
SerayeSVMNTAsn700ND
SerayeSVMNTAsn600ND
SerayeSVMNTAsn600ND
Kodondong AtasSVMNTAsn700ND
Kedondong AtasCVIETTyr450ND
Kedondong AtasCVIETTyr450ND
Kedondong AtasCVIETTyr450ND
Kedondong AtasSVMNTTyr900ND
Kedondong AtasCVIETTyr and Asn450ND
Apit AikSVMNTTyr and Asn750ND
Apit AikSVMNTAsn750ND
Apit AikSVMNTAsn800ND
Batu BolongSVMNTAsn750ND
Batu BolongSVMNTAsn650ND
Batu PenyuSVMNTTyr750ND
Batu PenyuSVMNTAsn750ND
Batu Penyu AtasSVMNTTyr750ND
Batu Penyu AtasSVMNTAsn750ND
Duduk AtasSVMNTTyr and Asn600ND
Duduk AtasSVMNTAsn950ND
Duduk AtasSVMNTTyr1000ND
Sd. KedondongSVMNTTyr and Asn700ND
Figure 1.
Figure 1.

Map of Indonesia showing the location of Lombok.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 1; 10.4269/ajtmh.2004.71.40

Authors’ addresses: Maria Cecilia Huaman, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan and Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mail: ceci_ huaman@yahoo.com. Kazumi Yoshinaga and Hiroji Kanbara, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan, Telephone: 81-95-849-7838, Fax: 81-95-849-7805. E-mail: f0512@cc.nagasaki-u.ac.jp. Aan Suryanatha and Nyoman Suarsana, Meninting Health Center, West Lombok, Indonesia.

Acknowledgments: We are grateful to the Case Detection and Treatment Team at the Meninting Health Center (West Lombok, Indonesia) for their collaboration in this study.

Financial support: This study was partially supported by the Japanese International Cooperation Agency partnership program, and by grant for International Health Cooperation Research (13-C-5) from the Ministry of Health, Labor and Welfare. Maria Cecilia Huaman is the recipient of the Monbusho scholarship awarded by the Ministry of Education, Science, Sports and Culture of the Government of Japan.

REFERENCES

  • 1

    Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LMB, Singh Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE, 2000. Mutations n 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
  • 2

    Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344 :257–263.

    • Search Google Scholar
    • Export Citation
  • 3

    Mehlotra RK, Fujioka H, Roepe PD, Janeeh O, Ursos LMB, Jacobs-Lorena V, McNamara DT, Bockarie MJ, Kazura JW, Kyle DE, Fidock DA, Zimmerman PA, 2001. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America. Proc Natl Acad Sci USA 98 :12689–12694.

    • Search Google Scholar
    • Export Citation
  • 4

    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
  • 5

    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
  • 6

    Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewsky C, Kawamoto F, Miller RS, Meshnick. 2003. Resistance to antimalarials in southeast Asia and genetic polymorphisms in pfmdr1.Antimicrobial Agents Chemother 47 :2418–2423.

    • Search Google Scholar
    • Export Citation
  • 7

    World Health Organization, 1990. The Clinical Management of Acute Malaria. Third edition. New Dehli: World Health Organization. Regional Publications, South East Asia Series No. 9.

  • 8

    Sumawinata IW, Bernadeta, Leksana B, Sutamihardja A, Purnomo, Subianto B, Sekartuti, Fryauff DJ, Baird JK. 2003. Very high risk of therapeutic failure with chloroquine for uncomplicated Plasmodium falciparum and Plasmodium vivax malaria in Indonesian Papua. Am J Trop Med Hyg 68 :416–420.

    • Search Google Scholar
    • Export Citation
  • 9

    Huaman MC, Roncal N, Nakazawa S, Long TTA, Gerena L, Garcia C, Solari L, Magill AJ, Kanbara H, 2004. Polymorphism of the Plasmodium falciparum multidrug resistance 1 and chloroquine resistance transporter genes and in vitro susceptibility to aminoquinolines in isolates from the Peruvian Amazon. Am J Trop Med Hyg 70 :1–6.

    • Search Google Scholar
    • Export Citation
  • 10

    Viriyakosol S, Siripoon N, Petcharapirat C, Petcharapirat P, Jarra W, Thaithong S, Brown KN, Snounou G, 1995. Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies. Bull World Health Organ 73 :85–95.

    • Search Google Scholar
    • Export Citation
  • 11

    Gomez-Saladin E, Fryauff DJ, Taylor WR, Laksana BS, Susanti AI, Purnomo, Subianto B, Richie TL. 1999. Plasmodium falciparum mdr1 mutations and in vivo chloroquine resistance in Indonesia. Am J Trop Med Hyg 61 :240–244.

    • Search Google Scholar
    • Export Citation
  • 12

    Nagesha HS, Din-Syafruddin, Casey GJ, Susanti AI, Fryauff DJ. 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 :43–49.

    • Search Google Scholar
    • Export Citation
  • 13

    Maguire DJ, Susanti AI, Krisin, Sismadi P, Fryauff DJ, Baird JK. 2001. The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia. Ann Trop Med Parasitol 95 :559–572.

    • Search Google Scholar
    • Export Citation
  • 14

    Plowe C, 2002. University of Maryland, Baltimore, MD. Web site: http://medschool.umaryland.edu/cvd/2002_pcr_asra.htm.

  • 15

    Cooper RA, Ferdig MT, Su XZ, Ursos LMB, Mu J, Nomura T, Fujioka H, Fidock DA, Roepe PD, Wellems TE, 2002. Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum.Mol Pharmacol 61 :35–42.

    • Search Google Scholar
    • Export Citation
  • 16

    Chen N, Russell B, Staley J, Kotecka B, Nasveld P, Cheng Q, 2001. Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum.J Infect Dis 183 :1543–1545.

    • Search Google Scholar
    • Export Citation
  • 17

    Chen N, Baker J, Ezard N, Burns M, Edstein MD, Cheng Q, 2002. Molecular evaluation of the efficacy of chloroquine treatment of uncomplicated Plasmodium falciparum malaria in East Timor. Am J Trop Med Hyg 67 :64–66.

    • Search Google Scholar
    • Export Citation
  • 18

    Lim P, Chy S, Ariey F, Incardona S, Chim P, Sem R, Denis MB, Hewitt S, Hoyer S, Socheat D, Merecreau-Puijalon O, Fandeur T, 2003. Pfcrt polymorphism and chloroquine resistance in Plasmodium falciparum strains isolated in Cambodia. Antimicrobial Agents Chemother 47 :87–94.

    • Search Google Scholar
    • Export Citation
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