Pyrosequencing primers Dylan Pillai, U of Calgary NEERI 26 November 2012 We thank Woodrow et al for their important commentary . Analysis of the pyrosequencing primers used for the detection of the two mutations in question (pfatp6 A623E and S769N), in this and a previous publication , confirmed the following. The Genbank ID (EF564342), which has also been reported by others , was used in the design of the pyrosequencing primers. However, this sequence is missing 14 amino acids at the N-terminus. As such, though the primers were designed to target the correct amino acid positions, they were based on the incomplete coding sequence and therefore did not identify the mutations in question. Hence, further Sanger-based sequencing will be required to confirm the presence or absence of mutations in PfATP6 sequences of these clinical isolates in relation to 50% inhibitory concentration (IC50) data. This work is to be undertaken. We note that a recent publication from Iran  identifies S769N mutations appearing after the introduction of artesunate, suggesting these mutations are still being identified in disparate geographic areas. 1. Woodrow CJ, Gardner KB and Bustamante LY. Questions over high frequency of mutant PfATP6 haplotypes in traveller isolates. Malaria Journal 2012, 11:186. 2. Shahinas D, Lau R, Khairnar K, Hancock D, Pillai DR. Artesunate misuse and Plasmodium falciparum malaria in traveler returning from Africa. Emerg Infect Dis. 2010 Oct;16(10):1608-10. 3. Kwansa-Bentum B,Ayi I, Suzuki T et al. Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro. Malaria Journal 2011, 10:187. 4. Zakeri S, Hemati S, Pirahmadi S, Afsharpad M, Raeisi A, Djadid ND. Molecular assessment of atpase6 mutations associated with artemisinin resistance among unexposed and exposed Plasmodium falciparum clinical isolates to artemisinin-based combination therapy. Malar J. 2012 Nov 9;11(1):373 Dr. Krishna Khairnar Environmental Health Division, National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur- 440020, Maharashtra India firstname.lastname@example.org Dr. Dylan Pillai Department of Pathology and Laboratory Medicine University of Calgary Canada Competing interests None declared..