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

Summary 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. Objectif Evaluer les sensibilité de base in vitroà l'artemether, à l'artésunate et à l'amodiaquine pour des souches de Plasmodium falciparum collectées en République démocratique de São Tomé et de Príncipe. Méthodes La prévalence de SNPs donnés dans les gènes Pfmdr1, le Pfcrt, le Pftctp et PfATPase6 a étéévaluée par PCR‐RFLP ou par séquençage d'ADN et le nombre de copies des gènes a été estimé par PCR à temps réel. Résultats Les concentrations inhibitrices 50 (CI50) moyennes pour l'artemether et l'artésunate étaient relativement basses (1,12 nm et 0,58 nm respectivement). Cependant, 10% des parasites ont démontré des valeurs de CI50 pour l'amodiaquine au delà du seuil de résistance accepté (60 nm) et il y avait une association positive pour la sensibilité entre les trois médicaments (artemether/artésunate: R = 0,84; artemether/l'amodiaquine: R = 0,68; artésunate /amodiaquine: R = 0,72). Les mutations dans les gènes Pfcrt et Pfmdr1étaient hautement prévalentes, tandis qu'un seul polymorphisme syn