Emergence of resistance to atovaquone-proguanil in malaria parasites: insights from computational modeling and clinical case reports

Abstract

The usefulness of atovaquone-proguanil (AP) as an antimalarial treatment is compromised by the emergence of atovaquone resistance during therapy. However, the origin of the parasite mitochondrial DNA (mtDNA) mutation conferring atovaquone resistance remains elusive. Here, we report a patient-based stochastic model that tracks the intrahost emergence of mutations in the multicopy mtDNA during the first erythrocytic parasite cycles leading to the malaria febrile episode. The effect of mtDNA copy number, mutation rate, mutation cost, and total parasite load on the mutant parasite load per patient was evaluated. Computer simulations showed that almost any infected patient carried, after four to seven erythrocytic cycles, de novo mutant parasites at low frequency, with varied frequencies of parasites carrying varied numbers of mutant mtDNA copies. A large interpatient variability in the size of this mutant reservoir was found; this variability was due to the different parameters tested but also to the relaxed replication and partitioning of mtDNA copies during mitosis. We also report seven clinical cases in which AP-resistant infections were treated by AP. These provided evidence that parasiticidal drug concentrations against AP-resistant parasites were transiently obtained within days after treatment initiation. Altogether, these results suggest that each patient carries new mtDNA mutant parasites that emerge before treatment but are killed by high starting drug concentrations. However, because the size of this mutant reservoir is highly variable from patient to patient, we propose that some patients fail to eliminate all of the mutant parasites, repeatedly producing de novo AP treatment failures.

FIG 1

Dynamics and loads of mtDNA mutant parasites per human host. The data are from 20,000 simulations, with one simulation representing one human infection. (A) Triangles represent homoplasmic-mutant parasites, and circles represent all-mutants (either heteroplasmic or homoplasmic-mutant); plain and dashed lines represent the median and the 99.5th upper percentile, respectively. (B) Triangles and circles represent homoplasmic-mutant and all-mutant parasites, respectively. (C and D) Data are from simulations stopped at the sixth erythrocytic 48-h cycle following parasite delivery from the liver (corresponding to a mean total parasite load per host of 1.5 × 10¹¹). The load of mutant parasites per host is shown, according to the number of mtDNA mutant copies per parasite. In panel C, each line (either plain or dashed) represents one simulated infected host randomly chosen among 20,000 simulations. Five simulations are shown. In panel D, box plots show the medians and interquartile ranges (IQR), and the bottom and top whiskers show the lowest data still within the lower quartile minus 1.5 times the IQR (1.5×IQR) and the highest data still within the upper quartile plus 1.5×IQR, respectively. Outlier values are shown as dots. There is no mutation cost, the mtDNA copy number is 20, and the mutation rate is 10⁻¹⁰/nucleotide/replication for all of the simulations presented here.

FIG 2

Load of mtDNA mutant parasites per human host according to different parameters. The data are from 20,000 simulations, with one simulation representing one infected host. (A, C, E, and G) All-mutant parasites, i.e., parasites having at least one of their n mtDNA copies mutated; (B, D, F, and H) homoplasmic-mutant parasites, i.e., parasites having all of their n mtDNA copies mutated. (E and F) At the fifth, sixth, and seventh erythrocytic 48-h cycles post liver, there were mean numbers of 1.2 × 10¹⁰, 1.5 × 10¹¹, and 1.8 × 10¹² parasites per host, respectively, in our simulations. Unless stated otherwise, the mutation rate was 10⁻¹⁰/nucleotide/replication, there was no resistance mutation cost (neutral), the mtDNA copy number was 20, and the parasite load per host was 1.5 × 10¹¹ (corresponding to the sixth erythrocytic 48-h cycle following parasite delivery from the liver). Abbreviations: neu, neutral; rec, recessive; dom, dominant. Box plots show the medians and IQR, and the bottom and top whiskers show the lowest data still within the lower quartile minus 1.5×IQR and the highest data still within the upper quartile plus 1.5×IQR, respectively. Outlier values are shown as dots.

FIG 3

Proportions of infections carrying mutant parasites. The data are from 20,000 simulations, with one simulation representing one human infected host. (A) The y axis scale ranges from 0 to 100; (B) same data as in panel A, but the y axis scale ranges from 0 to 2.5. Black, red, and blue lines represent infections after five, six, and seven erythrocytic 48-h cycles, respectively, following parasite release from the liver, corresponding to mean numbers of 1.2 × 10¹⁰, 1.5 × 10¹¹, and 1.8 × 10¹² parasites per host, respectively. The mtDNA copy number was 20, the mutation rate was 10⁻¹⁰/nucleotide/replication, and the mutation cost was recessive in the simulations presented here. A value of ≥1/20 refers to parasites with at least one of their 20 mtDNA copies mutated (i.e., all-mutant parasites), ≥10/20 refers to parasites with at least 10 of their 20 mtDNA copies mutated, and 20/20 refers to parasites with all of their 20 mtDNA copies mutated (i.e., homoplasmic-mutant parasites).