title: A modified in vitro sulfadoxine susceptibility assay for Plasmodium falciparum suitable for investigating Fansidar resistance.
abstract: The combination of pyrimethamine and sulfadoxine (PSD or Fansidar) represents one of the most important chemotherapeutic agents currently used to treat falciparum malaria. To investigate the molecular basis of resistance to PSD, reliable in vitro drug assays are required to permit correlation of resistance levels with different genotypes. We describe here protocols that permit accurate evaluation of IC50 values for sulfadoxine (SDX) inhibition of Plasmodium falciparum. Historically, tests for this drug have suffered from poor reproducibility and extreme variability in reported values. We have examined a series of variables, including serum-containing versus serum-free media, erythrocyte source, pre-test growth conditions, test components and post-test processing. We define conditions which better control the levels of the drug antagonists folate and p-aminobenzoate, yielding reproducible differences between lines of P. falciparum with differing alleles of the dihydropteroatic synthetase gene, which encodes the target enzyme of SDX. We also use this assay to demonstrate a marked difference in the response of different parasite lines to antagonism of SDX inhibition by exogenous folate. The ability to measure reliable IC50 values for SDX inhibition should greatly facilitate further experiments to explore the molecular basis of Fansidar resistance.
authors.affiliation: Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology (UMIST), UK.
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: P F
authors.initials: PF
authors.lastname: Sims
authors.forename: J E
authors.initials: JE
authors.lastname: Hyde
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000633:toxicity*; D016054:DNA, Protozoan / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004351:Drug Resistance; D004912:Erythrocytes / Q000469:parasitology; D005492:Folic Acid / Q000494:pharmacology; D006801:Humans; D019271:Hypoxanthine / Q000494:pharmacology; D010274:Parasitology / Q000379:methods; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods; D011739:Pyrimethamine / Q000633:toxicity*; D015203:Reproducibility of Results; D013413:Sulfadoxine / Q000633:toxicity*
chemical_list: D000962:Antimalarials; D016054:DNA, Protozoan; D004338:Drug Combinations; D019271:Hypoxanthine; C001205:fanasil, pyrimethamine drug combination; D013413:Sulfadoxine; D005492:Folic Acid; D011739:Pyrimethamine