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Background  Artemisinin-based combination therapy (ACT) is the mainstay of global efforts for treatment of  Plasmodium falciparum  malaria, but decline in its efficacy is the most important obstacle towards malaria control and elimination. Therefore, the present molecular analysis provides information on putative mutations associated with artemisinin resistance in  P .  falciparum  clinical population unexposed and exposed to artesunate 4 years after adoption of ACT as the first-line anti-malarial therapy in Iran.    Methods  In this study, blood samples (n = 226) were collected from uncomplicated  P .  falciparum -infected patients from different health centers of Chabahar district in Sistan and Baluchistan province in the south-eastern part of Iran, during 2003 to 2010. All collected isolates were analysed for putative candidate mutations (TTA) L263E (GAA), (GAA) E431K (AAA), (GCA) A623E (GAA) and (AGT) S769N (AAT) of  pfatpase6  gene using nested PCR/RFLP, followed by sequencing. Furthermore, the gene copy number was assessed by real-time quantitative PCR (RT-qPCR) in the presence of SYBR green.    Results  Neither the  pfatpase6  L263E nor the A623E mutation was detected among all examined isolates. The E431K mutation was found in 23% of the analysed samples unexposed to ACT; however, it was detected in 17.8% (34/191) of  P .  falciparum  isolates exposed to artesunate after 2007. High frequency of this single nucleotide polymorphisms (SNP) (overall 18.6%) among both examined groups ( X  2  test,  P >0.05) indicated that this SNP should be considered as an unrelated mutation to artemisinin resistance. In contrast, S769N mutation was not detected in unexposed isolates; however, it was found in 2.6% (5/191), four years after introduction of ACT in this malaria setting. Also, detected SNPs were not significantly frequent in both unexposed and exposed examined isolates ( X  2  test,  P > 0.05). Investigation in the copy number of  pfatpase6  gene revealed a similar number of copy (n = 1) as in an isolate sensitive to artemisinin.    Conclusion  Taken together, the results suggest, in particular, that  pfatpase6  S769N gene needs more consideration for its possible association with artesunate resistance among  P .  falciparum  isolates.

malaria journal

Molecular assessment of atpase6 mutations associated with artemisinin resistance among unexposed and exposed Plasmodium falciparum clinical isolates to artemisinin-based combination therapy