title: A high-throughput method for quantifying alleles and haplotypes of the malaria vaccine candidate Plasmodium falciparum merozoite surface protein-1 19 kDa.
abstract: BACKGROUND
  Malaria vaccine efficacy may be compromised if the frequency of non-target alleles increases following vaccination with a genetically polymorphic target. Methods are needed to monitor genetic diversity in polymorphic vaccine antigens, but determining which genetic variants of such antigens are present in infected individuals is complicated by the frequent occurrence of mixed infections.
  
  
  METHODS
  Pyrosequencing was used to determine allele frequencies at each of six single nucleotide polymorphisms in the Plasmodium falciparum blood-stage vaccine antigen merozoite surface protein 1 19 kDa (MSP-119) in field samples from a vaccine-testing site in Mali. Mixtures of MSP-119 clones were created to validate a haplotype-estimating algorithm that uses maximum likelihood methods to determine the most probable combination of haplotypes given the allele frequencies for an infection and the haplotypes known to be circulating in the population.
  
  
  RESULTS
  Fourteen unique MSP-119 haplotypes were identified among 351 genotyped infections. After adjustment to a standard curve, Pyrosequencing provided accurate and precise estimates of allele frequencies in mixed infections. The haplotype-estimating algorithm provided accurate estimates of haplotypes in mixed infections containing up to three haplotypes. Based on the MSP-119 locus, approximately 90% of the 351 infections contained two or fewer haplotypes.
  
  
  CONCLUSIONS
  Pyrosequencing in conjunction with a haplotype-estimating algorithm provides accurate estimates of haplotypes present in infections with up to 3 haplotypes, and can be used to monitor genetic diversity in parasite populations prior to and following introduction of MSP-1-based malaria vaccines.
authors.affiliation: Center for Vaccine Development, University of Maryland School of Medicine, HSF1-480, Baltimore, Maryland 21201, USA. stakala@medicine.umaryland.edu
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Takala
authors.forename: David L
authors.initials: DL
authors.lastname: Smith
authors.forename: O Colin
authors.initials: OC
authors.lastname: Stine
authors.forename: Drissa
authors.initials: D
authors.lastname: Coulibaly
authors.forename: Mahamadou A
authors.initials: MA
authors.lastname: Thera
authors.forename: Ogobara K
authors.initials: OK
authors.lastname: Doumbo
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Plowe
mesh_terms: D000328:Adult; D000465:Algorithms; D000483:Alleles; D000818:Animals; D016022:Case-Control Studies; D002648:Child; D011756:Diphosphates / Q000378:metabolism*; D005787:Gene Frequency; D006239:Haplotypes; D006801:Humans; D015994:Incidence; D017780:Malaria Vaccines; D016778:Malaria, Falciparum / Q000453:epidemiology / Q000517:prevention & control; D020066:Merozoite Surface Protein 1 / Q000235:genetics* / Q000276:immunology; D010963:Plasmodium falciparum / Q000145:classification / Q000235:genetics / Q000276:immunology; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D011756:Diphosphates; D017780:Malaria Vaccines; D020066:Merozoite Surface Protein 1