Plasmodium falciparum pfmdr1 amplification, mefloquine resistance, and parasite fitness

Abstract

Mefloquine is widely used in combination with artemisinin derivatives for the treatment of falciparum malaria. Mefloquine resistance in Plasmodium falciparum has been related to increased copy numbers of multidrug-resistant gene 1 (pfmdr1). We studied the ex vivo dynamics of pfmdr1 gene amplification in culture-adapted P. falciparum in relation to mefloquine resistance and parasite fitness. A Thai P. falciparum isolate (isolate TM036) was assessed by the use of multiple genetic markers as a single genotype. Resistance was selected by exposure to stepwise increasing concentrations of mefloquine up to 30 ng/ml in continuous culture. The pfmdr1 gene copy numbers increased as susceptibility to mefloquine declined (P = 0.03). No codon mutations at positions 86, 184, 1034, 1042, and 1246 in the pfmdr1 gene were detected. Two subclones of selected parasites (average copy numbers, 2.3 and 3.1, respectively) showed a fitness disadvantage when they were grown together with the original parasites containing a single pfmdr1 gene copy in the absence of mefloquine; the multiplication rates were 6.3% and 8.7% lower, respectively (P < 0.01). Modeling of the dynamics of the pfmdr1 copy numbers over time in relation to the relative fitness of the parasites suggested that net pfmdr1 gene amplification from one to two copies occurs once in every 10(8) parasites and that amplification from two to three copies occurs once in every 10(3) parasites. pfmdr1 gene amplification in P. falciparum is a frequent event and confers mefloquine resistance. Parasites with multiple copies of the pfmdr1 gene have decreased survival fitness in the absence of drug pressure.

FIG. 1.

Change in the median (range) pfmdr1 gene copy number of parasite strain TM036 over time in relation to exposure to increasing concentrations of mefloquine, as shown on the gray scale, from 15 ng/ml to 20 ng/ml, 25 ng/ml, and 30 ng/ml. Three observations were made at each time point. The checkered bars refer to culture in drug-free medium. *, P < 0.05 compared to the copy number at day 0; **, P < 0.05 compared to the copy number after the first exposure to the same mefloquine concentration.

FIG. 2.

Correlation between pfmdr1 copy number and mefloquine exposure.

FIG. 3.

Stability of pfmdr1 copy number genotype in drug-free medium over time in four parasite populations with different pfmdr1 copy numbers. □, control wild-type parasite (one copy); ▪, copy number after exposure to mefloquine at 15 ng/ml; ▴, copy number after exposure to mefloquine at 20 ng/ml; ○, 25 ng/ml; •, copy number after exposure to mefloquine at 30 ng/ml.

FIG. 4.

Change in average pfmdr1 copy number over time in cocultures of equal mixes of P. falciparum with a median of 1.0 and 2.3 copies (○) and 1.0 and 3.1 copy numbers (•). For comparison, the average copy numbers of a monoculture with one copy (▴), two copies (□), and three copies (▪) are also shown.