title: Molecular assessment of atpase6 mutations associated with artemisinin resistance among unexposed and exposed Plasmodium falciparum clinical isolates to artemisinin-based combination therapy.
abstract: 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 (X2 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 (X2 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Malaria and Vector Research Group MVRG, Biotechnology Research Center BRC, Pasteur Institut, P. O. Box 1316943551, Tehran, Iran. zakeris@yahoo.com
authors.forename: Sedigheh
authors.initials: S
authors.lastname: Zakeri
authors.forename: Samaneh
authors.initials: S
authors.lastname: Hemati
authors.forename: Sakineh
authors.initials: S
authors.lastname: Pirahmadi
authors.forename: Mandana
authors.initials: M
authors.lastname: Afsharpad
authors.forename: Ahmad
authors.initials: A
authors.lastname: Raeisi
authors.forename: Navid D
authors.initials: ND
authors.lastname: Djadid
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D000818:Animals; D000962:Antimalarials / Q000008:administration & dosage*; D037621:Artemisinins / Q000008:administration & dosage*; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004359:Drug Therapy, Combination; D018628:Gene Dosage; D017125:Genes, Protozoan; D006239:Haplotypes; D006801:Humans; D007492:Iran; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology*; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D016054:DNA, Protozoan; D015800:Protozoan Proteins; C031327:artemisinin; D000251:Adenosine Triphosphatases