title: Validation of the ligase detection reaction fluorescent microsphere assay for the detection of Plasmodium falciparum resistance mediating polymorphisms in Uganda.
abstract: BACKGROUND
  Malaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting.
  
  
  METHODS
  For 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared.
  
  
  RESULTS
  SNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K=0.77) for pfcrt K76T, 79% (K=0.55) for pfmdr1 N86Y, 83% (K=0.65) for pfmdr1 Y184F, and 91% (K=0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes.
  
  
  CONCLUSIONS
  The LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.
authors.forename: Sheila
authors.initials: S
authors.lastname: Nankoberanyi
authors.forename: George W
authors.initials: GW
authors.lastname: Mbogo
authors.forename: Norbert P
authors.initials: NP
authors.lastname: LeClair
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Conrad
authors.forename: Patrick
authors.initials: P
authors.lastname: Tumwebaze
authors.forename: Stephen
authors.initials: S
authors.lastname: Tukwasibwe
authors.forename: Moses R
authors.initials: MR
authors.lastname: Kamya
authors.forename: Jordan
authors.initials: J
authors.lastname: Tappero
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Nsobya
authors.affiliation: Department of Medicine, University of California, Box 0811, San Francisco, CA 94143, USA. prosenthal@medsfgh.ucsf.edu.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Rosenthal
mesh_terms: D002675:Child, Preschool; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004351:Drug Resistance; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D007223:Infant; D008025:Ligases / Q000378:metabolism; D008137:Longitudinal Studies; D016778:Malaria, Falciparum / Q000469:parasitology; D008297:Male; D008863:Microspheres; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D014454:Uganda
chemical_list: D016054:DNA, Protozoan; D008025:Ligases