title: In vitro assessment of artesunate, artemether and amodiaquine susceptibility and molecular analysis of putative resistance-associated mutations of Plasmodium falciparum from São Tomé and Príncipe.
abstract: OBJECTIVE
  To evaluate the basal in vitro responses of Plasmodium falciparum isolates collected in The Democratic Republic of São Tomé and Príncipe to artemether (ATH), artesunate (ATN) and amodiaquine (AMQ).
  
  
  METHODS
  The prevalence of given single nucleotide polymorphisms in the pfmdr1, pfcrt, pftctp and pfATPase6 genes was assessed by PCR-RFLP or DNA sequencing, and gene copy numbers were estimated by real-time PCR.
  
  
  RESULTS
  Mean IC50s to ATH and ATN were relatively low (1.12 nm and 0.58 nm, respectively). However, 10% of parasites displayed AMQ IC50 values above the accepted resistance threshold of 60 nm and there was a positive association between susceptibility to all three drugs (ATH vs. ATN: R = 0.84; ATH vs. AMQ: R = 0.68; ATN vs. AMQ: R = 0.72). Mutations in the pfcrt and pfmdr1 genes were highly prevalent, while only one synonymous polymorphism was detected in the pfATPase6 gene and no mutations were found in pftctp. All isolates harboured a single copy of the genes studied.
  
  
  CONCLUSIONS
  Artemisinin combination treatment in the São Tomé and Príncipe should be efficacious, although a significant number of AMQ-resistant parasites were detected and the susceptibility to each drug was positively associated with that of the other two. Mutations in the pfcrt and pfmdr1 genes are near fixation, most likely because of high levels of chloroquine resistance, whereas only one protein type of the artemisinin resistance candidate, PfATPase6, was identified.
authors.affiliation: Centro de Malária e Outras Doenças Tropicais/IHMT/UEI Biologia Molecular/UEI Malária, Lisbon, Portugal.
authors.forename: Isabel D
authors.initials: ID
authors.lastname: Ferreira
authors.forename: Dinora
authors.initials: D
authors.lastname: Lopes
authors.forename: Axel
authors.initials: A
authors.lastname: Martinelli
authors.forename: Conceição
authors.initials: C
authors.lastname: Ferreira
authors.forename: Virgílio E
authors.initials: VE
authors.lastname: do Rosário
authors.forename: Pedro
authors.initials: P
authors.lastname: Cravo
mesh_terms: D000655:Amodiaquine / Q000494:pharmacology*; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D000077549:Artemether; D037621:Artemisinins / Q000494:pharmacology*; D000077332:Artesunate; D001266:Atlantic Islands / Q000453:epidemiology; D002648:Child; D002675:Child, Preschool; D004351:Drug Resistance / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics; D006801:Humans; D016778:Malaria, Falciparum / Q000453:epidemiology; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012717:Sesquiterpenes / Q000494:pharmacology*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D012717:Sesquiterpenes; D000655:Amodiaquine; D000077332:Artesunate; D000077549:Artemether