Phenotypic Screens Reveal Posaconazole as a Rapidly Acting Amebicidal Combination Partner for Treatment of Primary Amoebic Meningoencephalitis

Abstract

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration-approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 μM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri-infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.

Keywords: Naegleria fowleri; azithromycin; drug discovery; miltefosine; phenotypic screen; posaconazole; primary amoebic meningoencephalitis.

Figure 1.

Rate of inhibition at 1 times the 50% inhibitory concentration (IC₅₀) for drugs currently used to treat primary amoebic meningoencephalitis (A), Food and Drug Administration–approved compounds identified through the St. Jude Children’s Research Hospital library screen (B), and known antifungals (C). Data are from a single experiment with 4 replicates but are representative of independent biological repeats (n = 3). RLU, relative luminescence unit.

Figure 2.

Isobolograms of posaconazole with azithromycin (A), miltefosine (B), or amphotericin B (C). The dotted line represents the line of additivity. Data are from a single experiment with 4 replicates but are representative of independent biological repeats (n = 3). FIC, fractional inhibitory concentration.

Figure 3.

Kaplan-Meier survival curves for 6 female ICR mice dosed intravenously with posaconazole at 20 mg/kg, 10 mg/kg, and 5 mg/kg; phosphate-buffered saline; or 20 mg/kg miltefosine. A survival rate of 33% was observed with mice treated with intravenous posaconazole (20 mg/kg; P < .05 by the log-rank test for trend).

Figure 4.

Survival curves for 10 female ICR mice infected with Naegleria fowleri trophozoites and dosed with azithromycin at 25 mg/kg (A), posaconazole at 20 mg/kg alone and in combination with azithromycin (B), fluconazole 30 mg/kg alone and in combination with azithromycin (C), and ketoconazole alone at 25 mg/kg and in combination with azithromycin (D). Solid lines represent drug alone, and dashed lines represent combination with azithromycin. Deaths are reported in half days.

Figure 5.

Fifty percent inhibitory concentration (IC₅₀) of multiple Centers for Disease Control and Prevention isolates and NF69 (reference strain). Each point represents 1 biological repeat with 2 replicates (n ≥ 3). Error bars represent standard errors of the mean.