Detecting adenosine triphosphatase 6 (PfATP6) point mutations that may be associated with Plasmodium falciparum   resistance to artemisinin: prevalence at baseline, before policy change in Uganda

The artemisinin based combination therapy (ACT) of artemether and lumefantrine (Co-artem) has recently replaced chloroquine and fansidar as the first line treatment policy drug in Uganda. It is necessary to develop practical procedures to monitor the likely emergence and spread of artemisinin resistant P. falciparum strains. We have analyzed the genotypes of PfATP6 in parasites from 300 stored filter paper samples from malaria patients who were diagnosed and treated in the years 1999 to 2004 at three field sites in Uganda. This is a period just prior to introduction of Co-artem. In order to develop a simple molecular procedure for mutation detection, regions of PfATP6 encoding protein domains important in artemisinin binding was amplified by nested PCR. Three DNA products, which together contain most of the coding region of amino acids located within the putative active site of pfATP6 were readily amplified. The amplified DNA was digested by restriction enzymes and the fragments sized by agarose gel electrophoresis. For the important codons 260, 263 and 769, methods using engineered restriction sites were employed. We did not find mutations at codons for the key residues Lys 260, Leu263, Gln266, Ser769 and Asn1039. Nucleotide sequencing of pfATPase6 gene DNA from at least 15 clinical isolates confirmed the above findings and suggested that mutations at these amino acid residues have not emerged in our study sites.