The evolutionary selection of malaria parasites within individual hosts is an important factor in the emergence of drug resistance but is still not well understood. We have examined the selection process for drug resistance in the mouse malaria agent Plasmodium berghei and compared the dynamics of the selection for atovaquone and pyrimethamine. Resistance to these drugs has been shown to be associated with genetic lesions in the dihydrofolate reductase gene in the case of pyrimethamine and in the mitochondrial cytochrome b gene for atovaquone. A mouse malaria model for the selection of drug resistance, based on repeated incomplete treatment (RICT) with a therapeutic dose of antimalarial drugs, was established. The number of treatment cycles for the development of stable resistance to atovaquone (2.47 ؎ 0.70; n ‫؍‬ 19) was found to be significantly lower than for pyrimethamine (5.44 ؎ 1.46; n ‫؍‬ 16; P < 0.0001), even when the parental P. berghei Leiden strain was cloned prior to the resistance selection. Similar results were obtained with P. berghei Edinburgh. Mutational changes underlying the resistance were identified to be S110N in dihydrofolate reductase for pyrimethamine and Y268N, Y268C, Y268S, L271V-K272R, and G280D in cytochrome b for atovaquone. These results are consistent with the rate of mitochondrial DNA mutation being higher than that in the nucleus and suggest that mutation leading to pyrimethamine resistance is not a rare event.D rug-resistant parasites have become a major challenge to malaria control today. The emergence of resistance is the outcome of two related processes: the genetic event that produces resistant mutants within individual hosts and the spread of resistance in populations. While, as the initial event in the emergence of resistance, the evolutionary selection of malaria parasites within an individual host is critical, it is still not well understood.The study of within-host selection of drug resistance benefits from animal models of malaria infection, as it allows genetic and physiological manipulations in vivo. Mutations can be selected without mosquito passage (i.e., without meiotic recombination) by exposure of large numbers of malaria parasites to certain drug concentrations. Drug-resistant Plasmodium falciparum organisms have been isolated in in vitro cultures (1-3), but drug-resistant Plasmodium berghei, Plasmodium yoelii, and Plasmodium chaubadi can be isolated in vivo in mice. Two general approaches to study in vivo antimalarial resistance selection have been employed and are compared by Peters (4): the serial technique (ST), in which drug dose is gradually increased after each passage, and the 2% relapse technique (2%RT), in which a single and high drug dose is administered at the time of each passage. While these approaches have proven to be effective in the selection of stable resistant strains, both are unnatural treatment regimens and thus might not be suitable models for the study of within-host emergence of antimalarial drug resistance.More recently, we have intr...

The evolutionary selection of malaria parasites within an individual host plays a critical role in the emergence of drug resistance. We have compared the selection of atovaquone resistance mutants in mouse models reflecting two different causes of failure of malaria treatment, an inadequate subtherapeutic dose and an incomplete therapeutic dose. The two models are based on cycles of insufficient treatment of Plasmodium berghei-infected mice: repeated inadequate treatment associated with a subtherapeutic dose (RIaT) (0.1 mg kg Ϫ1 of body weight) and repeated incomplete treatment with a therapeutic dose (RIcT) (14.4 mg kg Ϫ1 of body weight). The number of treatment cycles for the development of a stable resistance phenotype during RIaT was 2.00 Ϯ 0.00 cycles (n ϭ 9), which is not statistically different from that during RIcT (2.57 Ϯ 0.85 cycles; combined n ϭ 14; P ϭ 0.0591). All mutations underlying atovaquone resistance selected by RIaT (M133I, T142N, and L144S) were found to be in the Qo1 (quinone binding 1) domain of the mitochondrial cytochrome b gene, in contrast to those selected by RIcT (Y268N/C, L271V, K272R, and V284F) in the Qo2 domain or its neighboring sixth transmembrane region. Exposure of mixed populations of resistant parasites from RIaT to the higher therapeutic dose of RIcT revealed further insights into the dynamics of within-host selection of resistance to antimalarial drugs. These results suggest that both inadequate subtherapeutic doses and incomplete therapeutic doses in malaria treatment pose similar threats to the emergence of drug resistance. RIcT and RIaT could be developed as useful tools to predict the potential emergence of resistance to newly introduced and less-understood antimalarials.KEYWORDS dose-dependent selection, mouse malaria model, repeated inadequate treatment, repeated incomplete treatment, within-host selection of atovaquone resistance T he evolutionary selection of malaria parasites within an individual host plays a critical role in the emergence of antimalarial drug resistance, a major problem in malaria control. The study of within-host selection of drug resistance benefits from animal models of malaria infection, as it allows pharmacological manipulations in vivo.We recently reported such a model, based on cycles of repeated incomplete treatment (RIcT) of Plasmodium berghei-infected mice with a constant therapeutic dose of an antimalarial drug, which mimics treatment failure in the human field situation (1). We showed that under these conditions, stable resistance of P. berghei to the antimalarial atovaquone developed rapidly, after only 2.5 cycles of treatment.

BackgroundTo study within-host selection of resistant parasites, an important factor in the development of resistance to anti-malarial drugs, a mouse model of repeated interrupted malaria treatment (RIT) has been developed. The characteristics of within host selection of resistance to atovaquone and pyrimethamine in Plasmodium yoelii was examined in such a model.MethodsTreatment of P. yoelii infected mice, with atovaquone or pyrimethamine, was started at parasitaemia level of 3–5%, interrupted when reduced to less than 0.4%, and restarted following parasitaemia recovery to the initial level. Treatment cycles were repeated until stable phenotype resistance was observed.Results Plasmodium yoelii rapidly developed resistance to atovaquone (2.75 ± 1.06 cycles) and to pyrimethamine (5.4 ± 0.89 cycles) under RIT. A dose dependent phenomenon in the selection of atovaquone resistance mutations was observed. All mutations that underlie resistance to therapeutic doses of 0.3–1.44 mg kg−1 BW were found to be in the Qo2 domain of the cytochrome b gene (I258M, F267I/L/S, L271V, K272R, L271V and K272R). Those associated with lower doses of 0.01–0.03 mg kg−1 BW were in the Qo1 domain (M133I and T139S). The resistance mutations occurred at four of the 16 atovaquone putative drug binding sites suggested in P. falciparum. ConclusionsRIT of P. yoelii infected mice led to rapid development of resistance to atovaquone and pyrimethamine. The dose dependent selection of resistance mutants to atovaquone observed during RIT might reflect the outcome of two different causes of malaria treatment failure in human, repeated incomplete treatment with therapeutic dose and repeated inadequate treatment associated with sub-therapeutic dose, and need to be systematically investigated.