ACT is the mainstay of global efforts to control P. falciparum malaria, but suspected failure of artemisinin is the most important obstacle towards malaria control and elimination . At the present, no alternative classes of anti-malarial drugs are available to replace the artemisinin derivatives; hence, an urgent global priority is needed to prevent potentially the emergence and spread of artemisinin resistance. To achieve this goal, determining the molecular mechanism of artemisinin and its derivative resistance is very important. Therefore, the present molecular analysis provides, for the first time, baseline information on putative mutations (L263E, E431K, A623E and S769N) associated with artemisinin resistance in P. falciparum population unexposed and exposed to ACT in low endemic areas of Iran. In this study, by using nested PCR-RFLP followed by sequencing analysis, neither pfatpase6 L263E nor A623E mutations was detected among P. falciparum isolates unexposed (before 2007) and exposed (after 2007) to ACT. In addition, L263E mutation was never found in field isolates [3, 19, 20, 22, 23, 26, 29–33], similar to the present finding, and its association with artemisinin resistance needs more studies to be confirmed.
Concerning E431K, 23% of the isolates harbored this SNP in the collected samples before 2007; however, 17.8% were detected in P. falciparum samples exposed to artesunate after 2007. The pfatpase6 E431K mutation has been suggested to be associated with increased artesunate IC50 in Senegal  and this SNP has also been reported from African, Asian and South American P. falciparum isolates. In the present findings we found a high frequency of this SNP among P. falciparum population unexposed and exposed to artesunate, indicating that this SNP is unlikely associated with artemisinin resistance. Furthermore, the most prevalent haplotype was wild type with a frequency of 77% before 2007 and 80.6% after 2007. The second most frequent haplotype was L263
431A623S769 with 23% and 15.2% frequency before and after ACT adoption in Iran, respectively. In a previous in vitro study, this haplotype with single mutation was sensitive to dihydroartemisinin  and the results suggested that the high frequency of E431K among Cameroonian P. falciparum isolates might be a warning signal for artemisinin drug resistance. Also, although this mutation in high frequency (overall 18.6%) was found among Iranian P. falciparum isolates, the non-significant prevalence (X
2 test, P>0.05) among parasite population unexposed and exposed to ACT suggested that this mutation might have not been selected under artemisinin pressure.
Pfatpase6 S769N mutation has been reported to affect the PfSERCA activity with increased artemether IC50 and it was originally found in P. falciparum field isolates from French Guiana . However, it was not reported from China , Tanzania , Niger  and Brazil  with a different level of malaria endemicity. In parallel, this mutation was not detected in 35 Iranian P. falciparum isolates collected during 2001-2002 when CQ was used as first-line anti-malarial therapy . In addition, in the present investigation, further analysis on P. falciparum isolates collected during 2003-2006 (when CQ-SP was used as the first-line treatment) revealed no S769N mutation. Since ACT was used as the first-line treatment for uncomplicated falciparum malaria in Iran (in 2007), the frequency of this mutation has started to increase (2.6%). Detection of this mutation in only parasite isolates exposed to artesunate might suggest the likely selection of this mutation by artemisinin pressure as the previous study suggested the pfatpase6 S769N mutation as a potential molecular marker for P. falciparum resistance to artemether . Also, the most frequent haplotype before and after ACT adoption was wild type, but the number of haplotypes increased from 2 to 6 in parasite isolates unexposed and exposed to ACT, respectively (Notably, parasites carrying pfatpase6 S769N mutation). Nonetheless, recent report by Cui et al. , using allele exchange strategy revealed that 3D7 parasite carrying 769N mutation was still sensitive to artemisinin and its derivatives. This evaluation was done by in vitro response of transgenic lines to aforementioned drugs . Although this result argue against the predicted role of pfatpase 769N in resistance to artemisinin and its derivatives, but many variables including genetic backgrounds of the parasites may significantly influence on parasite resistance to various anti-malarial drugs [38–42]. Therefore, due to the conflicting data reported by different studies [19, 37], present study] the role of this candidate mutation in artemisinin and its derivative resistance needs further study.
In conclusion, the pfatpase6 genotype and its copy number might make early warning signals for the emergence of ACT resistance and provide baseline data for anti-malarial drug policy. Until now, there is no evidence for assuming that artemisinin resistance has occurred in Iran (Iranian Center for Disease Management and Control, surveillance report, unpublished). Therefore, the present results suggest that, to confirm and distinguish a mutation associated with artemisinin resistance, more studies are required among P. falciparum population unexposed and exposed to ACT from global malaria endemic setting. However, the pfatpase6 S769N mutation needs more awareness for its association with artesunate resistance and its value as a molecular marker for monitoring artemisinin resistance among P. falciparum population remains to be validated in areas where ACT has been used for a longer period. Introducing such a molecular tool could support the national and global malaria control and elimination programmes.