Within-Host Selection of Drug Resistance in a Mouse Model of Repeated Incomplete Malaria Treatment: Comparison between Atovaquone and Pyrimethamine

Abstract

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...

Within-Host Selection of Drug Resistance in a Mouse Model Reveals Dose-Dependent Selection of Atovaquone Resistance Mutations

“…The number of treatment cycles that led to parasite resistance to atovaquone in RIcT was found to be 2.40 Ϯ 0.89 cycles (mean Ϯ standard deviation [SD]), similar to the values in our previous report (2.67 Ϯ 0.87 cycles; P ϭ 0.5951) (1). 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 of RIcT (2.57 Ϯ 0.85 cycles; combined n ϭ 14; P ϭ 0.0591).…”

“…The new RIaT model is basically the same as the previously described RIcT model (1), except that the dose of atovaquone used for treatment was a subtherapeutic dose instead of a therapeutic dose. The number of treatment cycles for the development of a stable resistance phenotype during RIaT (a) is not statistically different from that for RIcT (b).…”

“…The development of the RIcT model was reported in detail previously (1). In this model, P. berghei-infected mice are intraperitoneally treated with a therapeutic dose of antimalarial drugs, which is interrupted every time the parasitemia level is reduced from 3 to 5% at the beginning of treatment to Ͻ0.4%, allowing the recovery of the parasitemia level before another treatment is administered.…”

“…Plasmodium berghei ANKA strain Leiden was obtained from Andy Waters, Leiden University Medical Center, Netherlands, and maintained by intraperitoneal passages in pathogen-free 10-to 12-week-old BALB/c mice (originally obtained from the Animal Resources Centre, Murdoch, Western Australia). The P. berghei stocks were screened for epizootic diarrhea of infant mice (EDIM), Mycoplasma pulmonis, mouse hepatitis virus (MHV), murine norovirus (MNV), mouse parvovirus (MPV), minute virus of mice (MVM), Theiler's encephalomyelitis virus (TMEV), and Sendai virus, as previously described (1). Approximately 10 6 parasitized red blood cells were inoculated per mouse at every passage.…”

“…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.…”