Volume 76, Issue 1
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


Individuals living in malaria endemic areas are often infected with multiple parasite clones. Currently used single nucleotide polymorphism (SNP) genotyping methods for malaria parasites are cumbersome; furthermore, few methods currently exist that can rapidly determine the most abundant clone in these complex infections. Here we describe an oligonucleotide ligation assay (OLA) to distinguish SNPs in the Duffy binding protein gene () at 14 polymorphic residues simultaneously. Allele abundance is determined by the highest mean fluorescent intensity of each allele. Using mixtures of plasmids encoding known haplotypes of the , single clones of parasites from infected monkeys, and well-defined mixed infections from field samples, we were able to identify the predominant genotype with > 93% accuracy when the dominant clone is twice as abundant as a lesser genotype and > 97% of the time if the ratio was 5:1 or greater. Thus, the OLA can accurately, reproducibly, and rapidly determine the predominant parasite haplotype in complex blood stage infections.


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  1. Cole-Tobian J, Biasor M, King CL, 2005. High complexity of Plasmodium vivax infections in Papua New Guinean children. Am J Trop Med Hyg 73 : 626–633. [Google Scholar]
  2. Hunt P, Fawcett R, Carter R, Walliker D, 2005. Estimating SNP proportions in populations of malaria parasites by sequencing: validation and applications. Mol Biochem Parasitol 143 : 173–182. [Google Scholar]
  3. McNamara DT, Kasehagen LJ, Grimberg BT, Cole-Tobian J, Collins WE, Zimmerman PA, 2006. Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent micro-sphere-based assay. Am J Trop Med Hyg 74 : 413–421. [Google Scholar]
  4. Li ZP, Kambara H, 2005. Single nucleotide polymorphism analysis based on minisequencing coupled with a fluorescence microsphere technology. J Nanosci Nanotechnol 5 : 1256–1260. [Google Scholar]
  5. Ye F, Li MS, Taylor JD, Nguyen Q, Colton HM, Casey WM, Wagner M, Weiner MP, Chen J, 2001. Fluorescent micro-sphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification. Hum Mutat 17 : 305–316. [Google Scholar]
  6. Dunbar SA, Vander Zee CA, Oliver KG, Karem KL, Jacobson JW, 2003. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex La-bMAP system. J Microbiol Methods 53 : 245–252. [Google Scholar]
  7. Cole-Tobian J, Cortes A, Baisor M, Kastens W, Xainli J, Bockarie M, Adams J, King C, 2002. Age-acquired immunity to a Plasmodium vivax invasion ligand, the Duffy binding protein. J Infect Dis 186 : 531–539. [Google Scholar]
  8. Tsuboi T, Kappe SH, Al-Yaman F, Prickett MD, Alpers M, Adams JH, 1994. Natural variation within the principal adhesion domain of the Plasmodium vivax Duffy binding protein. Infect Immun 62 : 5581–5586. [Google Scholar]
  9. Xainli J, Adams JH, King CL, 2000. The erythrocyte binding motif of Plasmodium vivax Duffy binding protein is highly polymorphic and functionally conserved in isolates from Papua New Guinea. Mol Biochem Parasitol 111 : 253–260. [Google Scholar]
  10. Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L, Medici MC, Arcangeletti MC, Snounou G, Dettori G, Chezzi C, 2004. Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 42 : 1214–1219. [Google Scholar]
  11. Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program fro windows 95/98/NT. Nucl. Acid Symp. Ser. 41 : 95–98. [Google Scholar]
  12. Fang XD, Kaslow DC, Adams JH, Miller LH, 1991. Cloning of the Plasmodium vivax Duffy receptor. Mol Biochem Parasitol 44 : 125–132. [Google Scholar]
  13. Kolakovich KA, Ssengoba A, Wojcik K, Tsuboi T, Al-Yaman F, Alpers M, Adams JH, 1996. Plasmodium vivax: favored gene frequencies of the merozoite surface protein-1 and the multiplicity of infection in a malaria endemic region. Exp Parasitol 83 : 11–18. [Google Scholar]
  14. Bruce MC, Galinski MR, Barnwell JW, Donnelly CA, Walmsley M, Alpers MP, Walliker D, Day KP, 2000. Genetic diversity and dynamics of Plasmodium falciparum and P. vivax populations in multiply infected children with asymptomatic malaria infections in Papua New Guinea. Parasitology 121 : 257–272. [Google Scholar]
  15. Kun JF, Missinou MA, Lell B, Sovric M, Knoop H, Bojowald B, Dangelmaier O, Kremsner PG, 2002. New emerging Plasmodium falciparum genotypes in children during the transition phase from asymptomatic parasitemia to malaria. Am J Trop Med Hyg 66 : 653–658. [Google Scholar]
  16. Genton B, Betuela I, Felger I, Al-Yaman F, Anders RF, Saul A, Rare L, Baisor M, Lorry K, Brown GV, Pye D, Irving DO, Smith TA, Beck HP, Alpers MP, 2002. A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea. J Infect Dis 185 : 820–827. [Google Scholar]
  17. Fluck C, Smith T, Beck HP, Irion A, Betuela I, Alpers MP, Anders R, Saul A, Genton B, Felger I, 2004. Strain-specific humoral response to a polymorphic malaria vaccine. Infect Immun 72 : 6300–6305. [Google Scholar]
  18. Ampudia E, Patarroyo MA, Patarroyo ME, Murillo LA, 1996. Genetic polymorphism of the Duffy receptor binding domain of Plasmodium vivax in Colombian wild isolates. Mol Biochem Parasitol 78 : 269–272. [Google Scholar]

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  • Received : 15 Mar 2006
  • Accepted : 13 Jul 2006

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