Competitive release and facilitation of drug-resistant parasites after therapeutic chemotherapy in a rodent malaria model

Abstract

Malaria infections frequently consist of mixtures of drug-resistant and drug-sensitive parasites. If crowding occurs, where clonal population densities are suppressed by the presence of coinfecting clones, removal of susceptible clones by drug treatment could allow resistant clones to expand into the newly vacated niche space within a host. Theoretical models show that, if such competitive release occurs, it can be a potent contributor to the strength of selection, greatly accelerating the rate at which resistance spreads in a population. A variety of correlational field data suggest that competitive release could occur in human malaria populations, but direct evidence cannot be ethically obtained from human infections. Here we show competitive release after pyrimethamine curative chemotherapy of acute infections of the rodent malaria Plasmodium chabaudi in laboratory mice. The expansion of resistant parasite numbers after treatment resulted in enhanced transmission-stage densities. After the elimination or near-elimination of sensitive parasites, the number of resistant parasites increased beyond that achieved when a competitor had never been present. Thus, a substantial competitive release occurred, markedly elevating the fitness advantages of drug resistance above those arising from survival alone. This finding may explain the rapid spread of drug resistance and the subsequently brief useful lifespans of some antimalarial drugs. In a second experiment, where subcurative chemotherapy was administered, the resistant clone was only partly released from competitive suppression and experienced a restriction in the size of its expansion after treatment. This finding raises the prospect of harnessing in-host ecology to slow the spread of drug resistance.

Fig. 1.

Parasite densities through time in experiment 1. Asexual density from qPCR (black lines) and gametocyte density from qRT-PCR (gray lines) are given for the resistant (a and b) and sensitive clone (c and d) in single (solid lines) and mixed (dotted lines) clone infections. Drug treatment (b and d) or sham injection (a and c) was administered on days 7–10 inclusively (marked by hashed vertical lines). Posttreatment sampling began on day 12. Minimum y axis value represents the lowest reliable detection threshold of qPCR. Mean densities (± 1 SEM) were calculated from all mice that were alive on the respective sampling day, a maximum of 20 per group at the start of the experiment. Malaria-induced deaths progressively reduced sample sizes, particularly in the untreated groups, and one to three mice were removed per group on days 6, 7, 13, and 14 for other experiments. Numbers of surviving mice during the key posttreatment phase are shown in Fig. 2.

Fig. 2.

Mean parasite density of drug-resistant clone in experiment 1. After 4 days of drug treatment, the resistant clone produced significantly more asexual parasites (a) and gametocytes (b) when the sensitive clone was present (dotted line) than when it was absent (solid line). Thus, elimination of the sensitive clone by chemotherapy resulted in a competitive release so great that the resistant clone performed better than it did in the prior absence of a coinfecting clone (treatment-dependent facilitation). Data points represent least squares mean (±1 SEM) of log-transformed total parasite density over days 12–14 (asexuals) and 13–14 (gametocytes) after infection for the mice surviving until the end of the sampling period. Numbers in brackets beside each point give the number of mice available for this analysis.

Fig. 3.

Asexual parasite (a–f) and gametocyte density (g–i) over the course of infection for experiment 2 determined by qPCR. The mean asexual densities (±1 SEM) of the resistant (a–c) and sensitive (d–f) clones in single (solid line) and mixed (dotted line) infections are shown for the 0-, 1-, and 2-day drug treatment groups. Only the gametocytes of the resistant clone are shown. Drug treatment began on day 7 after infection, as indicated by the hashed vertical lines; untreated mice received a sham injection. Posttreatment sampling began on day 11 after infection. The sensitive clone was suppressed by the subcurative chemotherapy, with higher drug dosage resulting in lower parasite densities (e and f). Two days of drug pressure and high reduction in the density of the sensitive clone resulted in the enhanced growth of the resistant clone in mixed infections (treatment-dependent facilitation) (c). One day of drug treatment resulted in competitive release but not enhanced growth of the resistant clone (b). Plotted points are a mean of mice surviving until day 21 (see Fig. 4). Absence of lines indicates samples undetected by qPCR.

Fig. 4.

Mean parasite density of drug-resistant clone in experiment 2. After 2 days of drug treatment, the resistant clone produced significantly more asexual parasites (a) and gametocytes (b) when in the presence of a competitor (dotted line) compared with the absence of a competitor (solid line), thus experiencing treatment-dependent facilitation. After 1 day of drug treatment, the resistant genotype still underwent competitive release, but it performed only as well as it did on its own. Points represent least squares mean (±1 SEM) of log-transformed total parasite density for days 12–21 after infection. Numbers in bracket show the numbers of mice surviving until the end of the sampling period, which were included in the analysis. Gametocytes were not detected by qRT-PCR in the single-infection 0- and 2-day drug treatment groups.