• View in gallery

    In vitro susceptibility to desethylamodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNPs are shadowed. NS; no sequence data.

  • View in gallery

    In vitro susceptibility to amodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNP are shadowed. NS; no sequence data.

  • 1

    World Health Organization, 2001. The Use of Antimalarial Drugs. Report of a WHO Informal Consulation. Geneva: Roll BacK Malaria/WHO.

  • 2

    Brasseur P, Guiguemde R, Diallo S, Guiyedi V, Kombila M, Ringwald P, Olliaro P, 1999. Amodiaquine remains effective for treating uncomplicated malaria in west and central Africa. Trans R Soc Trop Med Hyg 93 :645–650.

    • Search Google Scholar
    • Export Citation
  • 3

    Adjuik M BA, Garner P, Olliaro P, Taylor W, White N, International Artemisinin Study Group, 2004. Artesunate combinations for treatment of malaria: meta-analysis. Lancet 363 :9–17.

    • Search Google Scholar
    • Export Citation
  • 4

    Ministry of Health of Colombia, 1999. Guide for the Clinical Attention, Diagnosis and Treatment of Malaria. Santafé de Bogotá D.C.: General Direction of Health Promotion and Prevention.

  • 5

    Li XQ, Bjorkman A, Andersson TB, Ridderstrom M, Masimirembwa CM, 2002. Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate. J Pharmacol Exp Ther 300 :399–407.

    • Search Google Scholar
    • Export Citation
  • 6

    Churchill FC, Patchen LC, Campbell CC, Schwartz IK, Nguyen-Dinh P, Dickinson CM, 1985. Amodiaquine as a prodrug: importance of metabolite(s) in the antimalarial effect of amodiaquine in humans. Life Sci 36 :53–62.

    • Search Google Scholar
    • Export Citation
  • 7

    Hombhanje FW, Tsukahara T, Saruwatari J, Nakagawa M, Osawa H, Paniu MM, Takahashi N, Lum JK, Aumora B, Masta A, Sapuri M, Kobayakawa T, Kaneko A, Ishizaki T, 2004. The disposition of oral amodiaquine in Papua New Guinean children with falciparum malaria. Br J Clin Pharmacol 59 :1–4.

    • Search Google Scholar
    • Export Citation
  • 8

    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
  • 9

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

    • Search Google Scholar
    • Export Citation
  • 10

    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
  • 11

    Ochong EO, van den Broek IV, Keus K, Nzila A, 2003. Short report: association between chloroquine and amodiaquine resistance and allelic variation in the Plasmodium falciparum multiple drug resistance 1 gene and the chloroquine resistance transporter gene in isolates from the upper Nile in southern Sudan. Am J Trop Med Hyg 69 :184–187.

    • Search Google Scholar
    • Export Citation
  • 12

    Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Bjorkman A, 2006. Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y. Infect Genet Evol 6 :309–314.

    • Search Google Scholar
    • Export Citation
  • 13

    Happi CT, Gbotosho GO, Folarin OA, Bolaji OM, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AM, 2006. Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria. Am J Trop Med Hyg 75 :155–161.

    • Search Google Scholar
    • Export Citation
  • 14

    Gonzalez IJ, Padilla JO, Giraldo LE, Saravia NG, 2003. Efficacy of amodiaquine and sulfadoxine/pyrimethamine in the treatment of malaria not complicated by Plasmodium falciparum in Narino, Colombia, 1999–2002. Biomedica (Bogota) 23 :38–46.

    • Search Google Scholar
    • Export Citation
  • 15

    Blair SLL, Carmona-Fonseca J, Piñeros J, Ríos A, Alvarez T, Alvarez G, Tobón A, 2006. Therapeutic efficacy test in malaria falciparum in Antioquia, Colombia. Malar J 5 :14.

    • Search Google Scholar
    • Export Citation
  • 16

    Gonzalez IJ, Varela RE, Murillo C, Ferro BE, Salas J, Giraldo LE, Zalis MG, Saravia NG, 2003. Polymorphisms in cg2 and pfcrt genes and resistance to chloroquine and other antimalarials in vitro in Plasmodium falciparum isolates from Colombia. Trans R Soc Trop Med Hyg 97 :318–324.

    • Search Google Scholar
    • Export Citation
  • 17

    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.

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    Sidhu AB, Verdier-Pinard D, Fidock DA, 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 298 :210–213.

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    Mehlotra RK, Fujioka H, Roepe PD, Janneh O, Ursos LM, 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.

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    Cerutti Junior C, Marques C, Alencar FE, Durlacher RR, Alween A, Segurado AA, Pang LW, Zalis MG, 1999. Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method. Mem Inst Oswaldo Cruz 94 :803–809.

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    Vieira PP, Ferreira MU, Alecrim MG, Alecrim WD, da Silva LH, Sihuincha MM, Joy DA, Mu J, Su XZ, Zalis MG, 2004. pfcrt polymorphism and the spread of chloroquine resistance in Plasmodium falciparum populations across the Amazon Basin. J Infect Dis 190 :417–424.

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    Durrand V, Berry A, Sem R, Glaziou P, Beaudou J, Fandeur T, 2004. Variations in the sequence and expression of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and their relationship to chloroquine resistance in vitro. Mol Biochem Parasitol 136 :273–285.

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Polymorphisms in the pfcrt and pfmdr1 Genes of Plasmodium falciparum and in Vitro Susceptibility to Amodiaquine and Desethylamodiaquine

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  • 1 International Center for Medical Research and Training (CIDEIM), Cali, Colombia; Malaria Research Unit, Department of Infectious Diseases, Division of Medicine, Karolinska Institute, Stockholm, Sweden

The potential role of polymorphisms in the pfcrt and pfmdr1 genes and in vitro susceptibility to amodiaquine and desethylamodiaquine were explored in 15 chloroquine-resistant Colombian Plasmodium falciparum isolates. Single nucleotide polymorphisms in the pfcrt gene, including a newly reported mutation (S334N), were seen in isolates with decreased susceptibility to amodiaquine and desethylamodiaquine. The lowest susceptibility found to amodiaquine was observed in an isolate carrying a pfcrt and pfmdr1 Dd2-like haplotype, whereas a pfcrt haplotype related to the 7G8 Brazilian strain was found in a Colombian isolate with the lowest susceptibility to desethylamodiaquine. This exploratory study suggests that polymorphisms in the pfcrt and pfmdr1 genes play a role in amodiaquine and desethylamodiaquine resistance and warrants further study.

The development and expansion of resistance to the mainstay antimalarials chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) are the major causes for the increased morbidity and mortality of P. falciparum. In response to the increasing resistance, combination therapy, and especially artemisinin-based combination therapy (ACT), is now increasingly advocated as first-line therapy.1 The hypothesis is that a combination of drugs will achieve a more effective clinical and parasitologic cure, protect each other from the development of resistance, and reduce the overall rate of malaria. However, the choice of drugs is critical, especially bearing in mind the diversity and dynamics of P. falciparum and its possibility to develop different mechanisms of resistance.

The 4-aminoquinoline amodiaquine (AQ) is an antimalarial compound structurally and functionally related to CQ. It is more effective against CQ-resistant P. falciparum strains, and it is currently used in combination with artemisinin derivatives or SP for uncomplicated P. falciparum malaria, both in Africa2,3 and South America (Colombia).4 AQ is absorbed quickly in the gastrointestinal tract and metabolized in the liver by the cytochrome P450 isoform CYP2C8.5 Its main metabolite is desethylamodiaquine (DEAQ), which seems to exert the main antimalarial effect because of its significantly longer elimination half-life.6,7

Several polymorphisms in two P. falciparum genes, pfcrt and pfmdr1, have been associated with resistance to 4-aminoquinoline antimalarial drugs.810 Mainly pfcrt 76T, but also pfmdr1 86Y, has been involved in the development of CQ resistance and has also been associated with therapeutic failures after AQ treatment in Sudan, Kenya, and Nigeria.1113 In Colombia, where CQ-resistant P. falciparum is widespread but a more varied response to AQ has been found,14,15 the pfcrt 76T allele seems to be presently fixed and ubiquitous, whereas the pfmdr1 86Y allele seems to be rare.16,17

In vitro studies have shown that probably through the PFCRT protein, the Ca2+-transporter inhibitor drug verapamil (VP) may sensitize P. falciparum to CQ and to some extent to DEAQ but not to AQ.18,19 These differences between CQ and AQ/DEAQ and between AQ and DEAQ raise the hypothesis that other polymorphisms, maybe in the pfcrt or pfmdr1 genes, could mediate a decreased susceptibility to AQ and/or DEAQ.

To address this hypothesis and to further explore the resistance mechanisms against AQ/DEAQ, we analyzed the relationship of pfcrt and pfmdr1 mutations in 15 Colombian P. falciparum isolates with their in vitro susceptibility to CQ, AQ, and DEAQ. Documented in vitro data from four P. falciparum reference strains (Dd2, HB3, 3D7, and 7G8) were also included in the analysis as comparators.10,18,20

The 15 P. falciparum culture adapted isolates, collected between 1999 and 2001 in three endemic areas along the Colombian Pacific Coast, were analyzed in a radioisotopic in vitro susceptibility assay for their response to CQ, AQ, and DEAQ.21 The isolates were defrosted, adapted to continuous culture in supplemented RPMI-1640, and synchronized with sorbitol.2224 Ninety-six-well plates were coated with 50–3,200 nmol/L CQ and 5–320 nmol/L AQ and DEAQ after a 2-dilution factor (AQ and CQ were provided by the World Health Organization and DEAQ by the Walter Reed Army Institute). The final concentration of [3H] hypoxanthine was 0.5 μCi/well and that of Albumax I was 0.5%.21 The samples were incubated at 37°C for 48 hours, and each assay was done in duplicate. The counts per minute (cpm) were measured in a scintillation counter (LS7500; Beckman Instruments, Palo Alto, CA), and the IC50s were calculated using the PROBIT program in SPSS 7.5 for Windows 98 (SPSS, Chicago, IL).

The capacity of VP (V4629-1G; Sigma, St. Louis, MO) to sensitize P. falciparum to CQ, AQ, and DEAQ was tested in the isolates TA7519, CA2855, and Dd2 strain, according to the recently developed ELISA-histidine rich protein II (HRP2)–based assay.25 In vitro cultured parasites were diluted to an initial parasitemia of 0.05% and aliquoted into microculture 96-well plates pre-dosed with ascending concentrations of 16–2,020 nmol/L CQ, 2–270 nmol/L AQ, and 6–780 nmol/L DEAQ ± VP 0.8 μmol/L. After incubation at 37°C for 72 hours, the samples were freeze-thawed, transferred, and processed in pre-coated ELISA plates (Cellabs, New South Wales, Australia) for spectrophotometric analysis (Multiskan EX; Thermo Labsystems, Helsingfors, Finland) of parasite growth. The IC50 values were determined using HN-NonLin V1.05 Beta H. Noedl 2001 (http://malaria.farch.net). All samples were done in quadruple.

For the full sequencing of the pfcrt gene, total RNA and cDNA were obtained with Trizol LS (Invitrogen, Carlsbad, CA) and SUPERSCRIPT II RT (Invitrogen), respectively. Using the sequence of the HB3 strain (GenBank no. AF233068), primers were designed to amplify three overlapping fragments representing the complete pfcrt sequence (first block of 437 bp, second block of 409 bp, and third block of 593 bp). After a nested polymerase chain reaction (PCR) strategy, all blocks were sequenced (ABI 3700 Capillary DNA Sequencer; Cybergene AB, Huddinge, Sweden) and bioinformatically analyzed (Chromas V2.75, BLAST, Translate, LALING, and Clustal W). For the pfmdr1 single nucleotide polymorphism (SNP) analysis, a PCR-restriction fragment length polimorphism (RFLP) strategy26 was used to identify SNPs in positions N86Y, F184Y, S1034C, D1042N, and D1246Y (more details about these strategies can be requested from the authors). For the quantification of the pfmdr1 gene copy number, a TaqMan real-time PCR-based protocol (Applied Biosystems, Fresno, CA) was used as published elsewhere.27 Copy numbers were rounded to the nearest integer. This study was approved by the Ethical committees of CIDEIM and Universidad del Valle in Cali, Cali, Colombia.

In all 15 Colombian isolates, the IC50 values for CQ (114.58–415.53 nmol/L, data not shown) were above the WHO recommended cut-off for in vitro CQ resistance (IC50 >100 nmol/L). More variations in IC50 values were observed for both DEAQ (38.31–355.8 nmol/L; Figure 1) and AQ (9.63–64.61 nmol/L; Figure 2).

VP was able to considerably decrease the IC50 values in Dd2, CA2855, and TU7519 for CQ from 340.1 to 129.7; 636.9 to 182.3; and 551.7 to 70.9 nmol/L for each sample, respectively. Similarly, VP reduced the IC50 values for DEAQ from 32 to 12.8; 119.7 to 26.6; and 62.9 to 12.2 nmol/L, respectively. In contrast, VP did not modify the in vitro response to AQ: 13.1 to 11.0; 26.5 to 28.5; and 22.5 to 22.2 nmol/L, respectively.

DNA sequencing of the pfcrt gene was completely successful in 12 of 15 samples. Three samples were uncertain in the 371 position. A total of 15 SNPs in 12 positions in the pfcrt gene were identified (Figures 1 and 2). All 15 Colombian isolates carried the 76T and 220S mutant haplotype. In codons 72–76 of the pfcrt gene, the CVMET haplotype was seen in 13 of 15 isolates. The isolate TU741 carried a CVMNT haplotype, which has also been found in Ecuador and Peru.10,28 The isolate CA2855 showed a CVIET Dd2-like haplotype extending the reports of the presence of this Southeast Asian CQ-resistant haplotype in South America.10,28 The SVMNT haplotype characteristic of the 7G8 CQ-resistant strain from Brazil and reported in P. falciparum populations from Papua New Guinea,29 which has been proposed to be associated with DEAQ resistance,30 was not detected in any Colombian sample.

The SNP 97Q that to this date has been only reported in Colombia was found in the majority (13/15) of the parasites.10,17 In the TU741 isolate, a not previously described SNP, 334N, was identified. The SNP 333S was found in two isolates (TA4640 and TA6182); this SNP was previously found in Cambodia.31 In summary, we identified four pfcrt haplotypes among the fully sequenced samples (amino acids 72, 74–76, 97, 220, 271, 326, 333, 334, 356, and 371; SNPs are underlined): CMETQSQNTSIT (8/12), CMETQSQNSSII (2/12; GenBank no. DQ156109), CMNTHSQDTNLR (1/12; GenBank no. DQ156108), and CIETHSESTSTI (1/12).

Pfmdr1 analyses were successful in all Colombian isolates. Three pfmdr1 haplotypes (amino acids 86, 184, 1034, 1042, and 1246) were identified: NFSDD (6/15), NFSDY (8/15), and YYSND (1/15). All tested Colombian isolates had one pfmdr1 gene copy (Figures 1 and 2).

The high IC50 values for CQ and the more varying IC50 values for AQ and DEAQ, as well as the presence of pfcrt 76T and 220S in all Colombian samples, are in line with the high level of CQ resistance and the more various therapeutic response to AQ in Colombia. The VP results with sensitization for CQ and DEAQ but not for AQ confirm previously published data18,19 and suggest a mechanism of response to AQ less dependent on the pfcrt gene compared with DEAQ.

The TU741 isolate showed the highest IC50 value for DEAQ (355.8 nmol/L) and the second highest for AQ (41 nmol/L). TU741 was the only isolate with a pfcrt 72–76 CVMNT haplotype. TU741 also showed several genetic alterations not present in any of the other studied Colombian isolates, namely the pfcrt 326D and 356L SNPs also seen in the clone 7G8 (pfcrt 72–76 SVMNT), with the second highest IC50 value for DEAQ (229,3 nmol/L), as well as pfcrt 334N. This suggests that the haplotypes pfcrt 72–76 SVMNT/ CVMNT, 326D, 334N, and 356L might be associated with DEAQ resistance. The CA2855 isolate showed the highest IC50 for AQ (64.6 nmol/L) and the third highest to DEAQ (198.1 nmol/L). It was the only isolate carrying a Dd2-like SNP haplotype in both pfcrt and pfmdr1. However, CA2855 did have one copy of pfmdr1 compared with three in Dd2 (Figure 2). In addition, CA2855 was identified as FC27 and Dd2 as IC1 families in the msp2 gene (data not shown).

The presence of pfmdr1 86Y, 184Y, and 1042N in this isolate is consistent with recent in vivo data from Kenya where pfmdr1 86Y was selected among recrudescent infections but not among new re-infections after AQ monotherapy,12 suggesting that this haplotype is a potential factor for AQ resistance. However, Dd2 with the same haplotype had a lower IC50 value for AQ. Maybe this difference can be explained by its multicopied pfmdr1 gene or other genes involved in AQ resistance.

A visual analysis of the sequence data along with the IC50 values of DEAQ (Figure 1) and AQ (Figure 2) showed an accumulation of SNPs in the 3′ region of the pfcrt open reading frame (ORF). There was also a tendency of the pfmdr1 1246Y SNP toward higher IC50 values for AQ. A decrease in the hydrophobicity, volume of lateral chains of the amino acids, and changes in the charges of the PFCRT protein have been predicted to explain the mechanism by which these SNPs could decrease the efficacy of AQ.30

This exploratory study supports the hypothesis that AQ and DEAQ resistance might be associated with different combinations of polymorphisms in the pfcrt and pfmdr1 genes. The pfmdr1 copy number seems not be involved in 4-aminoquinolines resistance, which should be explored further.

Figure 1.
Figure 1.

In vitro susceptibility to desethylamodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNPs are shadowed. NS; no sequence data.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034

Figure 2.
Figure 2.

In vitro susceptibility to amodiaquine and genetic profile in the pfcrt and pfmdr1 genes of P. falciparum reference strains and Colombian isolates. SNP are shadowed. NS; no sequence data.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 6; 10.4269/ajtmh.2007.77.1034

*

Address correspondence to Diego F. Echeverry, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Valle, Colombia. E-mail: difereg@cideim.org.co

Authors’ addresses: Diego F. Echeverry, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Colombia, Telephone: 572–6682164, Fax: 572–6672989. Gabrielle Holmgren, Malaria Research Lab, M9/02, Karolinska Hospital, Stockholm 17176, Sweden. Claribel Murillo, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Valle, Colombia, Telephone: 572–6682164, Fax: 572–6672989. Juan C. Higuita, Universidad Nacional de Colombia, Manizales, Departamento de Ingeniería Química. Manizales, Colombia. Anders Bjorkman, Malaria Research Lab, M9/02, Karolinska Hospital, Stockholm 17176, Sweden. Jose P. Gil, Malaria Research Lab, M9/02, Karolinska Hospital, Stockholm 17176, Sweden. Lyda Osorio, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte 3–03, Cali, Colombia, Telephone: 572–6682164, Fax: 572–6672989.

Financial support: This work was supported by an anonymous Swiss foundation.

REFERENCES

  • 1

    World Health Organization, 2001. The Use of Antimalarial Drugs. Report of a WHO Informal Consulation. Geneva: Roll BacK Malaria/WHO.

  • 2

    Brasseur P, Guiguemde R, Diallo S, Guiyedi V, Kombila M, Ringwald P, Olliaro P, 1999. Amodiaquine remains effective for treating uncomplicated malaria in west and central Africa. Trans R Soc Trop Med Hyg 93 :645–650.

    • Search Google Scholar
    • Export Citation
  • 3

    Adjuik M BA, Garner P, Olliaro P, Taylor W, White N, International Artemisinin Study Group, 2004. Artesunate combinations for treatment of malaria: meta-analysis. Lancet 363 :9–17.

    • Search Google Scholar
    • Export Citation
  • 4

    Ministry of Health of Colombia, 1999. Guide for the Clinical Attention, Diagnosis and Treatment of Malaria. Santafé de Bogotá D.C.: General Direction of Health Promotion and Prevention.

  • 5

    Li XQ, Bjorkman A, Andersson TB, Ridderstrom M, Masimirembwa CM, 2002. Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate. J Pharmacol Exp Ther 300 :399–407.

    • Search Google Scholar
    • Export Citation
  • 6

    Churchill FC, Patchen LC, Campbell CC, Schwartz IK, Nguyen-Dinh P, Dickinson CM, 1985. Amodiaquine as a prodrug: importance of metabolite(s) in the antimalarial effect of amodiaquine in humans. Life Sci 36 :53–62.

    • Search Google Scholar
    • Export Citation
  • 7

    Hombhanje FW, Tsukahara T, Saruwatari J, Nakagawa M, Osawa H, Paniu MM, Takahashi N, Lum JK, Aumora B, Masta A, Sapuri M, Kobayakawa T, Kaneko A, Ishizaki T, 2004. The disposition of oral amodiaquine in Papua New Guinean children with falciparum malaria. Br J Clin Pharmacol 59 :1–4.

    • Search Google Scholar
    • Export Citation
  • 8

    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
  • 9

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

    • Search Google Scholar
    • Export Citation
  • 10

    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
  • 11

    Ochong EO, van den Broek IV, Keus K, Nzila A, 2003. Short report: association between chloroquine and amodiaquine resistance and allelic variation in the Plasmodium falciparum multiple drug resistance 1 gene and the chloroquine resistance transporter gene in isolates from the upper Nile in southern Sudan. Am J Trop Med Hyg 69 :184–187.

    • Search Google Scholar
    • Export Citation
  • 12

    Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Bjorkman A, 2006. Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y. Infect Genet Evol 6 :309–314.

    • Search Google Scholar
    • Export Citation
  • 13

    Happi CT, Gbotosho GO, Folarin OA, Bolaji OM, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AM, 2006. Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria. Am J Trop Med Hyg 75 :155–161.

    • Search Google Scholar
    • Export Citation
  • 14

    Gonzalez IJ, Padilla JO, Giraldo LE, Saravia NG, 2003. Efficacy of amodiaquine and sulfadoxine/pyrimethamine in the treatment of malaria not complicated by Plasmodium falciparum in Narino, Colombia, 1999–2002. Biomedica (Bogota) 23 :38–46.

    • Search Google Scholar
    • Export Citation
  • 15

    Blair SLL, Carmona-Fonseca J, Piñeros J, Ríos A, Alvarez T, Alvarez G, Tobón A, 2006. Therapeutic efficacy test in malaria falciparum in Antioquia, Colombia. Malar J 5 :14.

    • Search Google Scholar
    • Export Citation
  • 16

    Gonzalez IJ, Varela RE, Murillo C, Ferro BE, Salas J, Giraldo LE, Zalis MG, Saravia NG, 2003. Polymorphisms in cg2 and pfcrt genes and resistance to chloroquine and other antimalarials in vitro in Plasmodium falciparum isolates from Colombia. Trans R Soc Trop Med Hyg 97 :318–324.

    • Search Google Scholar
    • Export Citation
  • 17

    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
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