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VALIDATION OF MICROSATELLITE MARKERS FOR USE IN GENOTYPING POLYCLONAL PLASMODIUM FALCIPARUM INFECTIONS

Genotyping methods for Plasmodium falciparum drug efficacy trials have not been standardized and may fail to accurately distinguish recrudescence from new infection, especially in high transmission areas where polyclonal infections are common. We developed a simple method for genotyping using previously identified microsatellites and capillary electrophoresis, validated this method using mixtures of laboratory clones, and applied the method to field samples. Two microsatellite markers produced accurate results for single-clone but not polyclonal samples. Four other microsatellite markers were as sensitive as, and more specific than, commonly used genotyping techniques based on merozoite surface proteins 1 and 2. When applied to samples from 15 patients in Burkina Faso with recurrent parasitemia after treatment with sulphadoxine-pyrimethamine, the addition of these four microsatellite markers to msp1 and msp2 genotyping resulted in a reclassification of outcomes that strengthened the association between dhfr 59R, an anti-folate resistance mutation, and recrudescence (P = 0.31 versus P = 0.03). Four microsatellite markers performed well on polyclonal samples and may provide a valuable addition to genotyping for clinical drug efficacy studies in high transmission areas.

Received May 12, 2006. Accepted for publication July 12, 2006.

Acknowledgments: The authors thank Jiri Gut, Jennifer Legac, and Sarah Baxter for culturing parasites and performing flow cytometry.

Financial support: Support was provided from the National Institutes of Allergy and Infectious Disease (AI052142), Fogarty International Center/National Institutes of Health (TW00007), and the Doris Duke Charitable Foundation. P.J.R. is a Doris Duke Charitable Foundation Distinguished Clinical Scientist.

^* Address correspondence to Bryan Greenhouse, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143. E-mail: bgreenhouse{at}medsfgh.ucsf.edu

Authors’ addresses: Bryan Greenhouse, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143, Telephone: 415-206-8844, Fax: 415-648-8425, E-mail: bgreenhouse{at}medsfgh.ucsf.edu. Alissa Myrick, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143, Telephone: 415-206-3352, Fax: 415-648-8425, E-mail: amyrick{at}medsfgh.ucsf.edu. Christian Dokomajilar, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143, Telephone: 415-206-3349, Fax: 415-648-8425, E-mail: cdokomajilar{at}medsfgh.ucsf.edu. Jonathan Woo, Institute for Human Genetics, University of California, San Francisco, 401 Parnassus Avenue, Bldg. LPPI, Rm. LP70, Campus Box 0984, San Francisco, CA 94143, Telephone: 415-502-1576, Fax: 415-476-7389, E-mail: jonathan.woo{at}ucsf.edu. Elaine Carlson, Institute for Human Genetics, University of California, San Francisco, 401 Parnassus Avenue, Bldg. LPPI, Rm. LP70, Campus Box 0984, San Francisco, CA 94143, Telephone: 415-514-2289, Fax: 415-476-7389, E-mail: elaine.carlson{at}ucsf.edu. Philip J. Rosenthal, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143, Telephone: 415-206-8845, Fax: 415-648-8425, E-mail: prosenthal{at}medsfgh.ucsf.edu. Grant Dorsey, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143, Telephone: 415-206-4680, Fax: 415-648-8425, E-mail: gdorsey{at}medsfgh.ucsf.edu.

Reprint requests: Bryan Greenhouse, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143.

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