title: Multiple Origins of Mutations in the mdr1 Gene--A Putative Marker of Chloroquine Resistance in P. vivax.
abstract: BACKGROUND
  Chloroquine combined with primaquine has been the recommended antimalarial treatment of Plasmodium vivax malaria infections for six decades but the efficacy of this treatment regimen is threatened by chloroquine resistance (CQR). Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene, Pvmdr1 are putative determinants of CQR but the extent of their emergence at population level remains to be explored.
  
  
  OBJECTIVE
  In this study we describe the prevalence of SNPs in the Pvmdr1 among samples collected in seven P. vivax endemic countries and we looked for molecular evidence of drug selection by characterising polymorphism at microsatellite (MS) loci flanking the Pvmdr1 gene.
  
  
  METHODS
  We examined the prevalence of SNPs in the Pvmdr1 gene among 267 samples collected from Pakistan, Afghanistan, Sri Lanka, Nepal, Sudan, São Tomé and Ecuador. We measured and diversity in four microsatellite (MS) markers flanking the Pvmdr1 gene to look evidence of selection on mutant alleles.
  
  
  RESULTS
  SNP polymorphism in the Pvmdr1 gene was largely confined to codons T958M, Y976F and F1076L. Only 2.4% of samples were wildtype at all three codons (TYF, n = 5), 13.3% (n = 28) of the samples were single mutant MYF, 63.0% of samples (n = 133) were double mutant MYL, and 21.3% (n = 45) were triple mutant MFL. Clear geographic differences in the prevalence of these Pvmdr mutation combinations were observed. Significant linkage disequilibrium (LD) between Pvmdr1 and MS alleles was found in populations sampled in Ecuador, Nepal and Sri Lanka, while significant LD between Pvmdr1 and the combined 4 MS locus haplotype was only seen in Ecuador and Sri Lanka. When combining the 5 loci, high level diversity, measured as expected heterozygosity (He), was seen in the complete sample set (He = 0.99), while He estimates for individual loci ranged from 0.00-0.93. Although Pvmdr1 haplotypes were not consistently associated with specific flanking MS alleles, there was significant differentiation between geographic sites which could indicate directional selection through local drug pressure.
  
  
  CONCLUSIONS
  Our observations suggest that Pvmdr1 mutations emerged independently on multiple occasions even within the same population. In Sri Lanka population analysis at multiple sites showed evidence of local selection and geographical dispersal of Pvmdr1 mutations between sites.
authors.affiliation: Centre for Medical Parasitology, Institute of Immunology, and Microbiology, University of Copenhagen and Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Mette L
authors.initials: ML
authors.lastname: Schousboe
authors.affiliation: Centre for Medical Parasitology, Institute of Immunology, and Microbiology, University of Copenhagen and Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Samir
authors.initials: S
authors.lastname: Ranjitkar
authors.affiliation: Department of Zoology, University of Peradeniya, Sri Lanka.
authors.forename: Rupika S
authors.initials: RS
authors.lastname: Rajakaruna
authors.affiliation: International Water Management Institute, Hyderabad, Andhra Pradesh, India.
authors.forename: Priyanie H
authors.initials: PH
authors.lastname: Amerasinghe
authors.affiliation: Universidad San Francisco de Quito, Robles y Pampite, Cumbaya, Ecuador.
authors.forename: Francisco
authors.initials: F
authors.lastname: Morales
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Pearce
authors.affiliation: Blood-Borne Parasites, Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, United States of America.
authors.forename: Rosalyn
authors.initials: R
authors.lastname: Ord
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Toby
authors.initials: T
authors.lastname: Leslie
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Rowland
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Rockville, Maryland, United States of America.
authors.forename: Nahla B
authors.initials: NB
authors.lastname: Gadalla
authors.affiliation: Section of Global Health, Institute of Public Health, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Flemming
authors.initials: F
authors.lastname: Konradsen
authors.affiliation: Section of Global Health, Institute of Public Health, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Ib C
authors.initials: IC
authors.lastname: Bygbjerg
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Cally
authors.initials: C
authors.lastname: Roper
authors.affiliation: Centre for Medical Parasitology, Institute of Immunology, and Microbiology, University of Copenhagen and Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Michael
authors.initials: M
authors.lastname: Alifrangis
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D001208:Asia; D002738:Chloroquine / Q000494:pharmacology*; D004351:Drug Resistance; D004484:Ecuador; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009154:Mutation; D058974:Phylogeography; D010966:Plasmodium vivax / Q000187:drug effects* / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics*; D012641:Selection, Genetic; D013397:Sudan
chemical_list: D000962:Antimalarials; D002738:Chloroquine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; C541684:Mdr1 protein, Plasmodium vivax