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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 Plasmodium vivax Duffy binding protein gene (Pvdbp) 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 Pvdbp, single clones of P. vivax parasites from infected Aotus monkeys, and well-defined mixed infections from field samples, we were able to identify the predominant Pvdbp 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.
Received March 15, 2006. Accepted for publication July 13, 2006.
Acknowledgments: The authors thank William E. Collins at the Centers for Disease Control and Prevention for the parasitized monkey blood, and Dave McNamara for technical assistance and advice during the course of this study. We also thank the study participants from Papua New Guinea for their time.
Financial support: This study was supported by a grant from the Veteran’s Affairs Research Service. J.L.C.T. was supported by the ID/GeoMed Training Grant (T32-AI-07024) during this study.
* Address correspondence to Jennifer L. Cole-Tobian or Christopher L. King, Center for Global Health and Disease, Case Western Reserve University, Wolstein Research Building, Room 4132, 2103 Cornell Road, Cleveland, OH 44106. E-mail: jlc11{at}cwru.edu or christopher.king{at}case.edu.
Authors’ addresses: Jennifer L. Cole-Tobian and Christopher L. King, Case Western Reserve University, Center for Global Health and Disease, Wolstein Research Building, Room 4-132, 2103 Cornell Road, Cleveland, OH 44106-7286 and Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, E-mails: jlc11{at}cwru.edu and christopher.king{at}case.edu. Peter A. Zimmerman, Case Western Reserve University, Center for Global Health and Disease, Wolstein Research Building, Room 4-125, 2103 Cornell Road, Cleveland, OH 44106-7286, E-mail: paz{at}case.edu.
Reprint requests: Christopher L. King, Center for Global Health and Disease, Case Western Reserve University, Wolstein Research Building 4-132, 2103 Cornell Road, Cleveland, OH 44106.
This article has been cited by other articles:
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  C. L. King, P. Michon, A. R. Shakri, A. Marcotty, D. Stanisic, P. A. Zimmerman, J. L. Cole-Tobian, I. Mueller, and C. E. Chitnis Naturally acquired Duffy-binding protein-specific binding inhibitory antibodies confer protection from blood-stage Plasmodium vivax infection PNAS, June 17, 2008; 105(24): 8363 - 8368. [Abstract] [Full Text] [PDF]
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 J. L. Cole-Tobian, P. Michon, E. Dabod, I. Mueller, and C. L. King Dynamics of Asymptomatic Plasmodium vivax Infections and Duffy Binding Protein Polymorphisms in Relation to Parasitemia Levels in Papua New Guinean Children Am J Trop Med Hyg, November 1, 2007; 77(5): 955 - 962. [Abstract] [Full Text] [PDF]
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