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

BRYAN GREENHOUSE Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California; Genomics Core Facility, Institute for Human Genetics, University of California, San Francisco, California

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ALISSA MYRICK Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California; Genomics Core Facility, Institute for Human Genetics, University of California, San Francisco, California

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CHRISTIAN DOKOMAJILAR Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California; Genomics Core Facility, Institute for Human Genetics, University of California, San Francisco, California

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JONATHAN M. WOO Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California; Genomics Core Facility, Institute for Human Genetics, University of California, San Francisco, California

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ELAINE J. CARLSON Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California; Genomics Core Facility, Institute for Human Genetics, University of California, San Francisco, California

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PHILIP J. ROSENTHAL Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California; Genomics Core Facility, Institute for Human Genetics, University of California, San Francisco, California

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GRANT DORSEY Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California; Genomics Core Facility, Institute for Human Genetics, University of California, San Francisco, California

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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.

Author Notes

Reprint requests: Bryan Greenhouse, Department of Medicine, University of California, San Francisco, Box 0811, San Francisco, CA 94143.
  • 1

    Slater M, Kiggundu M, Dokomajilar C, Kamya MR, Bakyaita N, Talisuna A, Rosenthal PJ, Dorsey G, 2005. Distinguishing recrudescences from new infections in antimalarial clinical trials: Major impact of interpretation of genotyping results on estimates of drug efficacy. Am J Trop Med Hyg 73 :256–262.

    • Search Google Scholar
    • Export Citation
  • 2

    Snounou G, Beck HP, 1998. The use of PCR genotyping in the assessment of recrudescence or reinfection after antimalarial drug treatment. Parasitol Today 14 :462–467.

    • Search Google Scholar
    • Export Citation
  • 3

    Farnert A, Arez AP, Babiker HA, Beck HP, Benito A, Bjorkman A, Bruce MC, Conway DJ, Day KP, Henning L, Mercereau-Puijalon O, Ranford-Cartwright LC, Rubio JM, Snounou G, Walliker D, Zwetyenga J, do Rosario VE, 2001. Genotyping of Plasmodium falciparum infections by PCR: A comparative multicentre study. Trans R Soc Trop Med Hyg 95 :225–232.

    • Search Google Scholar
    • Export Citation
  • 4

    Anderson TJ, Haubold B, Williams JT, Estrada-Franco JG, Richardson L, Mollinedo R, Bockarie M, Mokili J, Mharakurwa S, French N, Whitworth J, Velez ID, Brockman AH, Nosten F, Ferreira MU, Day KP, 2000. Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum.Mol Biol Evol 17 :1467–1482.

    • Search Google Scholar
    • Export Citation
  • 5

    Su X, Wellems TE, 1996. Toward a high-resolution Plasmodium falciparum linkage map: polymorphic markers from hundreds of simple sequence repeats. Genomics 33 :430–444.

    • Search Google Scholar
    • Export Citation
  • 6

    Nyachieo A, Van Overmeir C, Laurent T, Dujardin JC, D’Alessandro U, 2005. Plasmodium falciparum genotyping by microsatellites as a method to distinguish between recrudescent and new infections. Am J Trop Med Hyg 73 :210–213.

    • Search Google Scholar
    • Export Citation
  • 7

    Mwangi JM, Omar SA, Ranford-Cartwright LC, 2006. Comparison of microsatellite and antigen-coding loci for differentiating recrudescing Plasmodium falciparum infections from reinfections in Kenya. Int J Parasitol 36 :329–336.

    • Search Google Scholar
    • Export Citation
  • 8

    Jensen JB, 2002. In vitro culture of Plasmodium parasites. Methods Mol Med 72 :477–488.

  • 9

    Musonda CC, Taylor D, Lehman J, Gut J, Rosenthal PJ, Chibale K, 2004. Application of multi-component reactions to antimalarial drug discovery. Part 1: Parallel synthesis and antiplasmodial activity of new 4-aminoquinoline Ugi adducts. Bioorg Med Chem Lett 14 :3901–3905.

    • Search Google Scholar
    • Export Citation
  • 10

    Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE, 1995. Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: Polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg 52 :565–568.

    • Search Google Scholar
    • Export Citation
  • 11

    Zwetyenga J, Rogier C, Tall A, Fontenille D, Snounou G, Trape JF, Mercereau-Puijalon O, 1998. No influence of age on infection complexity and allelic distribution in Plasmodium falciparum infections in Ndiop, a Senegalese village with seasonal, mesoendemic malaria. Am J Trop Med Hyg 59 :726–735.

    • Search Google Scholar
    • Export Citation
  • 12

    Cattamanchi A, Kyabayinze D, Hubbard A, Rosenthal PJ, Dorsey G, 2003. Distinguishing recrudescence from reinfection in a longitudinal antimalarial drug efficacy study: comparison of results based on genotyping of msp-1, msp-2, and glurp. Am J Trop Med Hyg 68 :133–139.

    • Search Google Scholar
    • Export Citation
  • 13

    Anderson TJ, Su XZ, Bockarie M, Lagog M, Day KP, 1999. Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples. Parasitology 119 :113–125.

    • Search Google Scholar
    • Export Citation
  • 14

    Rozen S, Skaletsky H, 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132 :365–386.

  • 15

    Brownstein MJ, Carpten JD, Smith JR, 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques 20 :1004–1006, 1008–1010.

    • Search Google Scholar
    • Export Citation
  • 16

    Shinde D, Lai Y, Sun F, Arnheim N, 2003. Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)n and (A/T)n microsatellites. Nucleic Acids Res 31 :974–980.

    • Search Google Scholar
    • Export Citation
  • 17

    Zongo I, Dorsey G, Rouamba N, Dokomajilar C, Lankoande M, Ouedraogo JB, Rosenthal PJ, 2005. Amodiaquine, sulfadoxine-pyrimethamine, and combination therapy for uncomplicated falciparum malaria: A randomized controlled trial from burkina faso. Am J Trop Med Hyg 73 :826–832.

    • Search Google Scholar
    • Export Citation
  • 18

    Duraisingh MT, Curtis J, Warhurst DC, 1998. Plasmodium falciparum: Detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp Parasitol 89 :1–8.

    • Search Google Scholar
    • Export Citation
  • 19

    Jafari S, Le Bras J, Bouchaud O, Durand R, 2004. Plasmodium falciparum clonal population dynamics during malaria treatment. J Infect Dis 189 :195–203.

    • Search Google Scholar
    • Export Citation
  • 20

    Walsh PS, Erlich HA, Higuchi R, 1992. Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods Appl 1 :241–250.

    • Search Google Scholar
    • Export Citation
  • 21

    Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, Brown KN, Viriyakosol S, 1999. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg 93 :369–374.

    • Search Google Scholar
    • Export Citation
  • 22

    Thompson JR, Marcelino LA, Polz MF, 2002. Heteroduplexes in mixed-template amplifications: Formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Res 30 :2083–2088.

    • Search Google Scholar
    • Export Citation
  • 23

    Tanabe K, Sakihama N, Farnert A, Rooth I, Bjorkman A, Walliker D, Ranford-Cartwright L, 2002. In vitro recombination during PCR of Plasmodium falciparum DNA: A potential pitfall in molecular population genetic analysis. Mol Biochem Parasitol 122 :211–216.

    • Search Google Scholar
    • Export Citation
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