Identification of the Plasmodium vivax mdr-Like Gene (pvmdr1) and ...

3 downloads 0 Views 522KB Size Report
Dec 9, 2004 - tially involved in drug resistance and of molecular markers related to drug resistance would provide a ..... Phillips EJ, Keystone JS, Kain KC.
MAJOR ARTICLE

Identification of the Plasmodium vivax mdr-Like Gene (pvmdr1) and Analysis of Single-Nucleotide Polymorphisms among Isolates from Different Areas of Endemicity Sara Brega,1 Benoit Meslin,1 Fre´de´rique de Monbrison,1,2 Carlo Severini,6 Luigi Gradoni,6 Rachanee Udomsangpetch,4 Inge Sutanto,5 Franc¸ois Peyron,1,3 and Ste´phane Picot1,2 1

Equipe d’Accueil 37-32, Parasitologie, Mycologie Me´dicale et Pathologie Exotique, Faculte´ de Me´decine, Universite´ Claude Bernard, and 2Service de Parasitologie, Mycologie Me´dicale et Maladies Tropicales, Hoˆpital E. Herriot, Hospices Civils de Lyon, and 3Service de Parasitologie et Maladies Tropicales, Hoˆpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; 4Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 5Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; 6 Department of Infectious, Parasitic, and Immunomediated Diseases, Istituto Superiore di Sanita`, Rome, Italy

Because of the lack of methods for continuous in vitro culture of Plasmodium vivax, little is known about drug-resistance mechanisms in this malaria-causing parasite. Therefore, identification of all the genes potentially involved in drug resistance and of molecular markers related to drug resistance would provide a framework for studying the incidence and spread of drug-resistant P. vivax strains. We have identified the P. vivax orthologue of the pfmdr1 gene (pvmdr1), which was shown to have a role in the drug resistance of Plasmodium falciparum. Comparison of the alignments of both nucleotide and amino acid sequences of pvmdr1 with those of other Plasmodium multidrug-resistance genes revealed an open-reading frame of 4392 base pairs encoding a deduced protein of 1464 amino acids. Nucleotide polymorphisms at 2 codons of the pvmdr1 gene—Y976F and F1076L—were found in 14 of 23 P. vivax isolates from different areas of endemicity, including Thailand, Indonesia, Turkey, Azerbaijan, and French Guyana. Plasmodium vivax causes more than half of all malaria infections outside Africa, with an estimated burden of ∼70–80 million cases/year [1]. In most areas of endemicity, chloroquine still constitutes the first-line therapy against uncomplicated malaria caused by P. vivax, followed by primaquine for eradication of asexual stages and hypnozoites. However, since 1989 (i.e., ∼30 years after chloroquine resistance [CQR] in Plasmodium falciparum was reported), the phenomenon of CQR in P. vivax has appeared in New Guinea and Sumatra [2], and there have been sporadic reports of CQR from

Received 7 June 2004; accepted 2 August 2004; electronically published 9 December 2004. Financial support: French Ministry of Research (programme PAL+, 2002); Re´gion Rhone-Alpes. Reprints or correspondence: Prof. Ste´phane Picot, Service de Parasitologie, Faculte´ de Me´decine, 8 Ave. Rockefeller, 69373 Lyon, Cedex 08, France (picot@ rockefeller.univ-lyon1.fr). The Journal of Infectious Diseases 2005; 191:272–7  2004 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2005/19102-0017$15.00

272 • JID 2005:191 (15 January) • Brega et al.

other geographic locations [3]. Two genes in P. falciparum, pfcrt [4] and pfmdr1 [5] (both coding for proteins associated to membrane transport), have been proposed to be involved in CQR. Field studies have confirmed that the pfcrt mutant allele K76T generally correlates with low clinical responses to the drug in patients with malaria and with parasite CQR detected in vitro [6–10]. Other studies have suggested that the mutant allele N86Y of the pfmdr1 gene may confer higher levels of resistance to parasites bearing the pfcrt mutant allele K76T [6, 11]. It has been demonstrated that CQR in P. vivax is not mediated by codon mutations in the pfcrt orthologue (pvcg10) [12]. Thus, it could be supposed that CQR mechanisms of P. vivax are different from those of P. falciparum. Of interest, the multidrug-resistance gene (mdr) shows orthologues in several Plasmodium species other than P. falciparum, including P. berghei [13], P. chabaudi [14], and P. yoelii [15]. In this report, we identify and

characterize an mdr-like gene (pvmdr1) in P. vivax isolates. We were interested to look for single-nucleotide polymorphisms (SNPs) in the sequence of this gene, to be used as molecular markers in the surveillance of CQR in P. vivax. Here, a relative high frequency of 2 SNPs in the pvmdr1 gene was found at codons not associated with CQR in P. falciparum. MATERIALS AND METHODS Parasite samples. Genomic P. chabaudi and P. yoelii DNA were a gift from Robert Menard and Paul Rick (Institut Pasteur de Paris, Paris). DNA from P. falciparum was obtained from culture-adapted strain 3D7, from The Netherlands, as described elsewhere [16]. After informed consent was obtained, 23 peripheral-blood samples were collected from patients with microscopically confirmed malaria caused by P. vivax, in different geographic locations, as follows: 9 blood samples were collected by fingerprick from adult patients visiting the Mae Sod malaria clinic, Tak province, Thailand; 3 blood-spot samples were collected during a malariometric survey in Alor district, East Nusa Tenggara province, Indonesia; 3 blood samples were collected by venipuncture into EDTA tubes from patients consulting at the Institute of Malariology of Adana in southeastern Turkey; 4 blood samples were collected into EDTA in central Azerbaijan during an active survey performed in the framework of a malaria project supported by the European Commission (INCO COPERNICUS-2 project [VIVAXNIS]); and 4 blood samples were collected into EDTA tubes from patients hospitalized in France who presented with P. vivax malaria imported from French Guyana. Preparation of DNA template from blood samples. Parasite genomic DNA from all blood samples collected into EDTA tubes was extracted by use of a QIAamp DNA blood kit (Qiagen), following the manufacturer’s instructions, with minor modifications: the incubation time with proteinase K was increased to 1 h at 56C, to improve the yield of the extraction, and DNA was eluted from the column by use of 100 mL of polymerase chain reaction (PCR)–grade H2O. DNA extraction of P. vivax blood-spot samples from Thailand and Indonesia was performed by use of the QIAamp DNA blood kit, following the Dried Blood Spots Protocol provided in the kit, with the same minor modifications described above. Identification of P. vivax mdr1 gene. Degenerate primers were designed to amplify within an mdr-like consensus sequence conserved between P. falciparum, P. chabaudi, and P. yoelii and were identified by a basic local alignment search tool (BLAST) search of National Center for Biotechnology information databases (available at: http://www.ncbi.nlm.nih.gov/ BLAST/). The degenerate oligonucleotide primers were pvmdrF2905 (forward; 5-CKGGTTTRGTAAATAATATTG-3) and pvmdr-4007 (reverse; 5-CATATTAAAYAACATKGGTTC-3), amplifying an expected fragment size of ∼1100 bp. Genomic

DNA from P. chabaudi, P. yoelii, P. falciparum, and a P. vivax isolate from Azerbaijan (Sab 2) were amplified in a total volume of 20 mL containing 0.20 mmol/L each dNTP, 1 mmol/L each primer, 1 U of Platinum Taq DNA Polymerase (Invitrogen), 1.5 mmol/L MgCl2, and 5 mL of 10⫻ PCR buffer. PCR was performed under the following conditions: 34 cycles of denaturation at 94C (2 min in the first cycle and 30 s in subsequent cycles), annealing for 1 min at 37C (during the first 5 cycles, the temperature between the annealing and extension phase increased to 68C, with a slope of 1C/4s), extension for 1.5 min at 68C, and a final primer extension for 5 min. Before sequencing of plasmid DNA, PCR products were separated on agarose gel, and the P. vivax product of expected size was gelpurified by use of a Geneclean turbo kit and was cloned by use of a TOPO TA Cloning kit (Invitrogen). BLAST searches to the P. vivax genome sequence data were performed at http:// PlasmoDB.org. The complete P. vivax sequence gene was retrieved in the chloroquine-sensitive (CQS) Sal-1 strain from El Salvador [17] and was submitted to GenBank under accession number AY618622. SYBR Green PCR amplification. Real-time PCR was performed using SYBR Green I dye binding specifically to doublestranded DNA. The sequences of PCR primers used to amplify 2 fragments (∼450 and 800 bp) from the P. vivax mdr1 gene of the Sal-1 strain (GenBank accession no. AY618622), corresponding to 2 polymorphic regions of pfmdr1 gene, were as follows: pvmdr1-F (5-CACCTTGCCCTTCTTCGT-3) and pvmdr1-R (5-TTCACCTTCTTGTTGCAAATAAC-3), and pvmdr1-S (5-ATAGTCATGCCCCAGGATTG-3) and pvmdr1AS (5-ACGTTTGGTCTGGACAAGTATC-3). Five microliters of DNA template was added to the PCR mixtures (20 mL) containing 0.5 mmol/L each primer, 3 mmol/ L MgCl2, and 2 mL of LightCycler FastStart DNA Master SYBR Green I buffer (Roche Molecular Biochemicals). The PCR program was performed for 40 cycles, as described elsewhere [18]. PCR product identity was assessed by a specific melting temperature obtained for each genotype. RESULTS Identification of the mdr-like gene in the P. vivax genome. In silico sequence analysis and PCR screening with codon-adjusted degenerate primers were used to identify P. vivax orthologues of P. falciparum (pfmdr1), P. chabaudi (pcmdr1), and P. yoelii (pymdr1). The ∼1100-bp P. vivax fragment amplified from the Sab 2 isolate was sequenced, and data analysis gave a significant match between part of the pfmdr1-coding sequence (GenBank accession no. M29154), as well as pcmdr1 and pymdr1 sequences (GenBank accession nos. AY123625 and PY00245, respectively). Consensus prediction of the translated partial amino acid sequence confirmed the homology with PfMDR1 and showed the presence of 4 hydrophobic transmembrane domains (segP. vivax mdr-Like Gene • JID 2005:191 (15 January) • 273

Figure 1. Comparison of the predicted sequences for pfmdr1 (GenBank accession no. M29154) and its homologues pvmdr1 (GenBank accession no. AY618622) and pcmdr1 (GenBank accession no. AY123625). Matching amino acids across all species are marked as follows: bold dots (.) indicate weak similarity, bold colons (:) indicate strong similarity, and bold asterisks (*) indicate identity between corresponding amino acids. Boxes with solid lines indicate putative transmembrane segments, and boxes with dashed lines enclose the 2 nucleotide-binding consensus sequences (NBS1 and NBS2). The Walker A and Walker B motifs are in shaded boxes. Bold dashes (–) representing spaces have been inserted to optimize the comparisons.

Table 1. Mutations present in the pvmdr1 gene amplified from Plasmodium vivax isolates. Mutation residues in pvmdr1 amino acid Isolate/location

91

189

976

1076

Guy1/French Guyana Guy2/French Guyana Guy3/French Guyana TMPV108/Thailand TMPV109/Thailand VP1439/Thailand A33/Indonesia VP1438/Thailand Sal-1/El Salvadora 84/French Guyana VP1443/Thailandb VP1445/Thailand F49/Indonesia Mi17/Indonesia VP1448/Thailand VP1454/Thailand Tur11/Turkey Agy6/Azerbaijan VP1447/Thailand Tur22/Turkey Tur3/Turkey Sab5/Azerbaijan Saa12/Azerbaijan Sab2/Azerbaijanc

N N N N N N N N N N N N N N N N N N N N N N N N

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Y Y Y Y Y Y Y Y Y Y F F F F F F Y Y Y Y Y Y Y Y

F F F F F F F F F F L L L L L L L L L L L L L L

NOTE. Residues that differ from the wild-type Sal-1 strain are indicated in bold type. Representative sequences were submitted to Genbank. a b c

GenBank accession no. AY618622 (wild-type strain). GenBank accession no. AY643799. GenBank accession no. AY351844.

ments IX, X, XI, and XII) and 1 highly conserved nucleotidebinding site (NBS1) of the pfmdr1 protein. The sequence data reported from P. vivax mdr-like gene have been submitted to GenBank and assigned the accession number AY351844. The complete putative mdr-like P. vivax sequence gene (GenBank accession no. AY618622) was characterized in a monkey-adapted parasite isolate, the CQS Sal-1 strain [17], retrieved from PlasmoDB (available at: http://PlasmoDB.org). Comparison of this presumed nucleotide sequence of the pvmdr1 gene with the P. falciparum orthologue, pfmdr1, revealed a single open-reading frame (ORF) of 4392 bp encoding a deduced protein of 1464 aa. The initiator methionine is located at position 1, and there is a continuous coding region to a stop codon at position 1464. The guanine and cytosine content of the sequence is 42%, a figure typical for P. vivax–coding sequences [19]. Comparative alignments of the amino acid sequences deduced from pvmdr1, pcmdr1, and pfmdr1 genes (figure 1) showed striking conservation in overall composition and structural features. The identity rate between the 3 coding se-

quences is 56.8% (68.33% with P. falciparum alone), and that between the deduced amino acid sequences is 61.37% (75.48% with P. falciparum alone). The transmembrane prediction obtained by the mean of the Kyte-Doolittle hydropathy values indicates that the PvMDR1 protein has 12 transmembrane segments conserved in spacing and orientation. Motif searches showed the presence of typical signal sequences of ATP-binding motif in each half of the molecule. Analysis of SNPs in the pvmdr1 gene by use of SYBR Green. DNA from 23 P. vivax–infected samples yielded a mean (SD) specific melting temperature of 82.1  0.24 C for the first pvmdr1 fragment (450 bp) and 84.9  0.36C for the second pvmdr1 fragment (800 bp) amplified with SYBR Green I. All pvmdr1 sequences were confirmed by direct sequencing of both strands of the purified PCR products. Sequencing results of the first fragment, encompassing codon positions 91 and 189 (which are homologous to the polymorphic sites 86 and 184 in P. falciparum, respectively), showed the absence of nonsynonymous mutations among the 23 isolates analyzed. The only unexpected synonymous mutation observed was found at codon 173 (GGCrGGT) in 2 Turkish samples. Sequencing results of the second pvmdr1 fragment, encompassing codon positions 1071 and 1079 (which are homologous to the polymorphic sites 1034 and 1042 in P. falciparum, respectively), showed 2 nonsynonymous mutations, at positions 976 and 1076 (table 1). The YrF change at codon 976 (TACrTTC) was observed in 6 (26%) of 23 samples: 4 samples from Thailand and 2 from Indonesia. The second FrL change (at codon 1076), which was due to a single mutation (TTTrCTT), was observed in 14 (61%) of 23 samples: 5 from Thailand (4 of them bearing the mutation at codon 976), 4 from Azerbaijan, 3 from Turkey, and 2 from Indonesia (the latter being the same isolates bearing the mutation at codon 976). DISCUSSION The phenomenon of CQR in P. vivax has been extensively described in Papua New Guinea in 1989 [2] and in Indonesia in 1991 [20, 21]; more recently, sporadic clinical CQR has been reported in India [22, 23], Myanmar [24], and in Central and South America [25–27]. However, the mechanisms of CQR in P. vivax are still unknown. Although the action of chloroquine is probably similar in P. vivax and in P. falciparum, it appears that the development of CQR involves a different mechanism in these 2 species [12]. Codon mutations in the pfcrt gene are central to the CQR phenotype of P. falciparum, but P. vivax pvcg10, the chloroquine-resistant transporter gene (crt) orthologue of P. falciparum, has been shown to be not responsible for CQR in P. vivax [12]. P. vivax mdr-Like Gene • JID 2005:191 (15 January) • 275

The P. vivax mdr gene has not yet been described, probably because the role of the orthologue in P. falciparum (pfmdr1) is less important than that of pfcrt in CQR. However, identification of an orthologue between the 2 major Plasmodium species is important for cross-species comparison of gene function and comparison of molecular mechanisms associated with a common phenotype, such as CQR [28]. In the present article, we have reported the first evidence of a P. vivax mdr-like gene and have described some polymorphisms in isolates from areas where CQR in P. vivax has been detected, such as Indonesia. The pvmdr1 gene is characterized by a single ORF of 4392 bp, encoding a protein of 1464 aa. Two regions of the pvmdr1 gene have been taken into consideration: one encompassing the P. vivax positions homologous to the polymorphic sites 86 and 184 in P. falciparum, and the other encompassing the P. vivax positions homologous to the polymorphic sites 1034 and 1042 in P. falciparum. Sequence analysis of the pvmdr1 gene has shown no polymorphism at pvmdr1 codon 91 homologous to pfmdr1 codon 86, which is associated with drug resistance in P. falciparum. These data contrast with those obtained for pyrimethamineresistant malaria parasites, in which mutations in dihydrofolate reductase gene, the target enzyme for this folate drug, are at similar sites in the genome of both Plasmodium species [18]. Amplification or overexpression of the mdr1 genes from various Plasmodium species have also been associated with drug resistance, but the level of expression of the pvmdr1 gene and the copy number of this gene have not been determined for these samples. Two pvmdr1 mutant alleles were identified: F1076L alone and Y976F-F1076L. The locations of the 2 mutations in the MDR protein are the X and XI hydrophobic segments, respectively. The rates of distribution of pvmdr1 genotypes found among our samples were 42% for the wild-type genotype, 33% for the single-mutant genotype, and 25% for the double-mutant genotype. Unfortunately, the role of these mutations in clinical CQR has not been evaluated in patients infected with mutant Plasmodium species. However, assessment of the therapeutic failures are confounded by the tendency of P. vivax malaria to relapse from liver hypnozoites several weeks after initial infection, and, in areas of endemicity where continuous malaria transmission occurs, it is often difficult to distinguish between reinfection, relapse, and recrudescence [29–31]. Thus, we are still unable to assume that these polymorphisms could be related to any drug resistance, and the same polymorphisms have not been described yet for P. berghei, P. chabaudi, and P. yoelii. Nevertheless, the high frequency of these mutations in samples from different areas of endemicity and from different continents is probably not without significance. It should be noted that all the double mutants (976–1076) were found in samples from Thailand and Indonesia. In the latter area, CQR in P. 276 • JID 2005:191 (15 January) • Brega et al.

vivax has been reported for more than a decade. Other cases of CQR in P. vivax have been reported in Papua New Guinea [2], India [22, 23], and Myanmar [24], which all are Asian countries. Thus, even if most of the P. vivax infections detected in Thailand still seem to be CQS, we could speculate that these 2 mutations could be early molecular markers of P. vivax CQR and should be included in later surveys of drug resistance. A potential use of real-time PCR will be the analysis of a large number of samples in a short period of time. On the other hand, only some samples from Azerbaijan and Turkey had a single mutation at position 1076, and none had a double mutation. This result is concordant with the good efficacy rate of chloroquine against P. vivax in these areas [32]. Surprisingly, no mutation has been observed in samples from French Guyana, where CQR in P. vivax has been reported. However, the very low number of samples studied from each area led us to analyze these data with great care. Further studies are needed to explore the value of these new molecular markers in the context of increasing P. vivax drug resistance.

Acknowledgments We thank Daniel Schmitt for continued support.

References 1. Mendis K, Sina BJ, Marchesini P, Carter R. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 2001; 64:97–106. 2. Rieckmann KH, Davis DR, Hutton DC. Plasmodium vivax resistance to chloroquine? Lancet 1989; 2:1183–4. 3. Baird JK, Leksana B, Masbar S, et al. Diagnosis of resistance to chloroquine by Plasmodium vivax: timing of recurrence and whole blood chloroquine levels. Am J Trop Med Hyg 1997; 56:621–6. 4. Fidock DA, Nomura T, Talley AK, et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 2000; 6:861–71. 5. Foote SJ, Thompson JK, Cowman AF, Kemp DJ. Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum. Cell 1989; 57:921–30. 6. Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D. 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 2001; 183:1535–8. 7. Chen N, Russell B, Staley J, Kotecka B, Nasveld P, Cheng Q. Sequence polymorphisms in pfcrt are strongly associated with chloroquine resistance in Plasmodium falciparum. J Infect Dis 2001; 183:1543–5. 8. Djimde A, Doumbo OK, Cortese JF, et al. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 2001; 344: 257–63. 9. Dorsey G, Kamya MR, Singh A, Rosenthal PJ. Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr-1 genes and clinical response to chloroquine in Kampala, Uganda. J Infect Dis 2001; 183:1417–20. 10. Mayor AG, Gomez-Olive X, Aponte JJ, et al. Prevalence of the K76T mutation in the putative Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene and its relation to chloroquine resistance in Mozambique. J Infect Dis 2001; 183:1413–6. 11. Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum

12.

13.

14.

15.

16. 17.

18.

19. 20.

21.

is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol 2000; 108:13–23. Nomura T, Carlton JMR, Baird K, et al. Evidence for different mechanisms of chloroquine resistance in 2 Plasmodium species that cause human malaria. J Infect Dis 2001; 183:1653–61. Gervais GW, Trujillo K, Robinson BL, Peters W, Serrano AE. Plasmodium berghei: identification of an mdr-like gene associated with drug resistance. Exp Parasitol 1999; 91:86–92. Hunt P, Cravo PVL, Donleavy P, Carlton JM-R, Walliker D. Chloroquine-resistance in Plasmodium chabaudi: are chloroquine-resistance transporter (crt) and multidrug resistance (mdr1) orthologues involved? Mol Biochem Parasitol 2004; 133:27–35. Ferrer-Rodriguez I, Perez-Rosado J, Gervais GW, Peters W, Robinson BL, Serrano AE. Plasmodium yoelii: identification and partial characterization of an MDR1 gene in an artemisinin-resistant line. J Parasitol 2004; 90:152–60. Walliker D, Quakyi IA, Wellems TE, et al. Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 1987; 236:1661–6. Contacos PG, Collins WE, Jeffery GM, Krotoski WA, Howard WA. Studies on the characterization of Plasmodium vivax strains from Central America. Am J Trop Med Hyg 1972; 21:707–12. Brega S, de Monbrison F, Severini C, et al. Real-time PCR for dihydrofolate reductase gene single-nucleotide polymorphisms in Plasmodium vivax isolates. Antimicrob Agents Chemother 2004; 48:2581–7. Carlton J. The Plasmodium vivax genome sequencing project. Trends Parasitol 2003; 19:227–31. Baird JK, Basri H, Subianto B, Patchen LC, Hoffman SL. Resistance to chloroquine by Plasmodium vivax in Irian Jaya, Indonesia. Am J Trop Med Hyg 1991; 44:547–52. Schwartz IK, Lackrtiz EM, Patchen LC. Chloroquine resistance Plasmodium vivax from Indonesia [letter]. New Engl J Med 1991; 324:927.

22. Garg M, Gopinathan N, Bodhe P, Khirsagar NA. Vivax malaria resistant to chloroquine: case reports from Bombay. Trans R Soc Trop Med Hyg 1995; 89:656–7. 23. Dua VK, Kar PK, Sharma VP. Chloroquine resistant Plasmodium vivax malaria in India. Trop Med Int Health 1996; 1:816–9. 24. Kyaw MP, Kyaw MP, Myint O, et al. Emergence of chloroquine-resistant Plasmodium vivax in Myanmar (Burma) [case report]. Trans R Soc Trop Med Hyg 1993; 87:687. 25. Phillips EJ, Keystone JS, Kain KC. Failure of combined chloroquine and high-dose primaquine therapy for Plasmodium vivax malaria acquired in Guyana, South America. Clin Infect Dis 1996; 23:1171–3. 26. Soto J, Toledo J, Gutierrez P, et al. Plasmodium vivax clinically resistant to chloroquine in Colombia. Am J Trop Med Hyg 2001; 65:90–3. 27. Alecrim das G, Alecrim W, Macedo V. Plasmodium vivax resistance to chloroquine (R2) and mefloquine (R3) in Brazilian Amazon region. Rev Soc Bras Med Trop 1999; 32:67–8. 28. Carlton JMR, Muller R, Yowell CA, et al. Profiling the malaria genome: a gene survey of three species of malaria parasite with comparison to other apicomplexan species. Mol Biochem Parasitol 2001; 118:201–10. 29. Whitby M. Drug resistant Plasmodium vivax malaria. J Antimicrob Chemother 1997; 40:749–52. 30. Pukrittayakamee S, Imwong M, Looareesuwan S, White NJ. Therapeutic responses to antimalarial and antibacterial drugs in vivax malaria. Acta Trop 2004; 89:351–6. 31. Sattabongkot J, Tsuboi T, Zollner GE, Sirichaisinthop J, Cui L. Plasmodium vivax transmission: chances for control? Trends Parasitol 2004; 20:192–8. 32. Valibayov A, Abdullayev F, Mammadov S, et al. Clinical efficacy of chloroquine followed by primaquine for Plasmodium vivax treatment in Azerbaijan. Acta Trop 2003; 88:99–102.

P. vivax mdr-Like Gene • JID 2005:191 (15 January) • 277