In vitro assessment of artesunate, artemether and amodiaquine susceptibility and molecular analysis of putative resistance-associated mutations of Plasmodium falciparum from Sao Tome and Principe

OBJECTIVE To evaluate the basal in vitro responses of Plasmodium falciparum isolates collected in The Democratic Republic of Sao Tome and Principe to artemether (ATH), artesunate (ATN) and amodiaquine (AMQ). METHODS The prevalence of given single nucleotide polymorphisms in the pfmdr1, pfcrt, pftctp and pfATPase6 genes 1 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 Sao Tome and Principe 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.