Amphotericin B and fluconazole, a potent combination therapy for cryptococcal meningitis

Abstract

We evaluated the antifungal activities of amphotericin B, fluconazole, and flucytosine, alone and in combination, in a murine model of cryptococcal meningitis. The objectives were to determine the greatest antifungal effects achievable with these drugs alone or in combination. Meningitis was established in male BALB/c mice weighing 23 to 25 g by intracerebral injection of Cryptococcus neoformans. Treatment was started on day 2. Amphotericin B was tested at 0.3 to 1.3 mg/kg of body weight/day by slow intravenous injection. Fluconazole at 10 to 40 mg/kg/day and flucytosine at 20 to 105 mg/kg/day were administered in the sole source of drinking water. The mice were killed at 16 days, and the numbers of fungal colonies in the brain were quantified. The association between the response and the dose combination was evaluated by local nonparametric response surface methods; 99% confidence intervals were used to evaluate the antifungal effects. Ninety-five percent of the mice treated with amphotericin B at 0.5 mg/kg survived to the end of the experiment, regardless of the fluconazole or flucytosine dose used. The greatest activity was seen with amphotericin B plus fluconazole with or without flucytosine. However, the addition of flucytosine did not increase the antifungal activity. Given the widespread availability of amphotericin B and fluconazole and the relative safety profile of fluconazole compared to that of flucytosine, the full potential of this two-drug combination deserves further evaluation.

FIG. 1.

In vitro susceptibility of C. neoformans isolate USC 1597 to amphotericin B (A), fluconazole (B), and flucytosine (C). The curves show the loess fits of the association between drug concentration and the numbers of CFU per milliliter (log₁₀) after 48 h of incubation. Vertical lines above and below the curves show the 99% CIs for log₁₀ CFU per milliliter.

FIG. 2.

Proportion of deaths within the first 24 h of administration of the first dose of amphotericin B by dose level.

FIG. 3.

Numbers of CFU of C. neoformans per gram of brain tissue by day postinfection for untreated control animals. The symbols at day 0 represent the median inoculum level (log₁₀ CFU per gram of brain tissue) in experiments 1 and 2.

FIG. 4.

Response surfaces showing the association between numbers of CFU per gram of brain tissue and dose combinations of fluconazole (Flu) with or without flucytosine (5FC) and without amphotericin B (AmB) (A) and fluconazole with or without flucytosine and with amphotericin B (B). The color key shows the levels of response (numbers of log₁₀ CFU per gram of brain tissue) associated with shades of pink.

FIG. 5.

Dose-response curves for fluconazole (Flu) without flucytosine (5FC) (open symbols) and for flucytosine at 105 mg/kg/day (filled symbols) without amphotericin B (AmB) (A) and with amphotericin B (B). The lines above and below the symbols represent the 99% CIs for the numbers of CFU per gram of brain. Horizontal references lines are shown at 2, 4, 6, and 8 log₁₀ CFU/g to facilitate comparison of CIs among the dose regimens.

FIG. 6.

Contour plots of the upper limits of the 99% CIs for the numbers of CFU per gram of brain tissue for fluconazole with or without flucytosine and without amphotericin B (A) and with amphotericin B (B). The contours and shades of pink identify dose combinations with similar levels of response. The color key shows the CI level (numbers of log₁₀ CFU per gram of brain tissue) associated with each shade of pink.

FIG. 7.

Response for ABCD at 5 mg/kg/day alone, ABCD with flucytosine (5FC) at 100 mg/kg, and ABCD with flucytosine and fluconazole (Flu). The lines above and below the symbols represent the 99% CIs for the numbers of CFU per gram of brain tissue. Horizontal references lines are shown at 2, 4, 6, and 8 log₁₀ CFU/g to facilitate comparison of CIs. Data are from Diamond et al. (9).