Malaria parasites giving rise to recrudescence in vitro

Abstract

Recrudescences were simulated in vitro with drug treatment to examine how drug-sensitive parasites survive the treatment. Various numbers of cultured parasites were treated with lethal doses of pyrimethamine or mefloquine for various lengths of time. Recrudescences were observed in parasite populations with larger initial numbers of parasites when the treatment duration was prolonged. Equal numbers of parasitized erythrocytes were treated with various concentrations of pyrimethamine or mefloquine. There was no clear linear relationship between the incidence of recrudescence and the drug concentration. Parasites that had recrudesced were continuously allowed to recrudesce in the succeeding recrudescence experiments. Both the duration from the cessation of treatment to the time at which the recrudescent parasitemia level reached 1% and the growth rate of recrudescent parasites were equal among these recrudescences. The recrudescent parasites in these experiments were as sensitive to the drugs as the parasites tested before treatment were. These results suggest that a parasite culture may contain parasites in some phases that are not killed by drug for up to 10 days, which explains the recrudescences that occur even after treatment.

FIG. 1.

Recrudescent parasites showed the same patterns of sensitivity to pyrimethamine (PYR) as the control parasites did. (A) Control parasites; (B to H) recrudescent parasites: 10⁵ for 4 days (B), 10⁶ for 4 days (C), 10⁶ for 6 days (D), 10⁷ for 6 days (E), 10⁸ for 8 days (F), 10⁹ for 8 days (G), and 10⁹ for 10 days (H).

FIG. 2.

Recrudescent parasites showed the same pattern of sensitivity to mefloquine (MEF) as the control parasites did. (A) Control parasites; (B to E) recrudescent parasites: 10⁶ for 4 days (B), 10⁷ for 4 days (C), 10⁷ for 6 days (D), 10⁸ for 6 days (E).

FIG. 3.

(A) Parasites recrudesce independently of the pyrimethamine concentration. Fifty million parasitized erythrocytes (7% parasitemia) were exposed to the indicated concentrations of pyrimethamine for 4 days, washed, and returned to standard medium. Experiments were carried out in triplicate. Mean ± standard deviation levels of parasitemia and mean ± standard deviation durations from the day when treatment ceased until the day when the recrudescent parasitemia level reached or surpassed 1% are shown. The data are representative of three separate experiments. Means followed by the same letter were not significantly different (P = 0.744). (B) Recrudescent parasites showed the same pattern of sensitivity to pyrimethamine (PYR) as the parasites tested before treatment did.

FIG. 4.

(A) Parasites recrudesce independently of the mefloquine concentration. Fifty million parasitized erythrocytes (6.5% parasitemia) were treated with the indicated concentrations of mefloquine. Experiments were carried out in triplicate. Mean ± standard deviation levels of parasitemia and mean ± standard deviation durations from the day when treatment ceased until the day when the recrudescent parasitemia level reached or surpassed 1% are shown. Data shown are representative of three separate experiments. Means followed by the same letter were not significantly different (P = 0.565). (B) Recrudescent parasites showed the same pattern of sensitivity to mefloquine (MEF) as the parasites tested before treatment did.

FIG. 5.

Residual mefloquine suppressed parasite growth, and its effect was reduced during the incubation. Parasitized erythrocytes at 18% parasitemia were diluted with fresh erythrocytes that had been treated as follows: by treatment with 10⁻⁶ M mefloquine for 4 days and washing (filled circles); by treatment with mefloquine, washing, and incubation in standard medium for 7 days (open triangles); or by incubation in standard medium (open circles). Parasite culture was carried out in triplicate. Mean ± standard deviation levels of parasitemia are shown. The data are representative of two separate experiments.

FIG. 6.

Recrudescent parasites showed the same pattern of sensitivity to pyrimethamine (PYR) as parasites tested before treatment did.

FIG. 7.

Killing by pyrimethamine treatment and growth of recrudescent parasites. The figure was prepared with the data from Table 1. All pyrimethamine treatment durations except the 6-day treatment against 10⁶ parasites eradicated within the treatment duration. Broken lines, killing rate if the killing rate remains constant with succeeding cycles; dotted lines, numbers of surviving parasites extrapolated from a growth rate above the detection limit.