Assessment of pfmdr1 gene copy number by tandem competitive polymerase chain reaction

Abstract

The pfmdr1 gene encodes a Plasmodium falciparum homologue of the human P-glycoprotein expressed on the surface of the parasite food vacuole. Variation in copy number and specific codon mutations of pfmdr1 have been implicated in the development of parasite resistance to antimalarial drugs. We describe here the technique of Tandem-Competitive Polymerase Chain Reaction (TC-PCR), which allows accurate measurement of pfmdr1 copy number in parasite DNA obtained directly from small quantities (100 μl) of red blood cells. We reliably quantified pfmdr1 in previously well characterised strains of Plasmodium falciparum with differing pfmdr1 gene copy numbers using starting amounts of between 3000 and 40000 gene copies. We then used TC-PCR to determine pfmdr1 gene copy number in field specimens of venous blood taken from 10 patients with malaria contracted along the Thai-Burmese border. In this region of high grade parasite resistance to mefloquine greater than 70% of samples had a copy number greater than 1 of pfmdr1 determined with a repeatability coefficient of 0.58.