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

Sedigheh Zakeri1*, Samaneh Hemati2, Sakineh Pirahmadi1, Mandana Afsharpad1, Ahmad Raeisi34 and Navid D Djadid1

Author Affiliations

1 Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institut, P.O. Box 1316943551, Tehran, Iran

2 Biology Department, Khatam University, Tehran, Iran

3 National Programme for Malaria Control, Ministry of Health and Medical Education, Tehran, Iran

4 Tehran University of Medical Sciences, School of Public Health, Tehran, Iran

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Malaria Journal 2012, 11:373  doi:10.1186/1475-2875-11-373

Published: 9 November 2012

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.

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.