Introduction

Mycetoma is a serious health problem with high morbidity. It is endemic in subtropical areas and often leads to severe deformity and disability [1]. The disease has long been disregarded by international health organizations but was recently recognized by WHO as a neglected tropical condition (http://www.who.int/neglected_diseases/diseases/en/). One of the main problems of eumycetoma is its recalcitrant nature, which necessitates prolonged antifungal therapy combined with massive and repeated surgical debridement. In severe cases, amputation of the affected part may be the only remaining treatment option [2]. Madurella mycetomatis is the most common fungal pathogen causing eumycetoma in arid climate zones, particularly in northeastern Africa. The infection by M. mycetomatis is characterized by the presence of black grains in tissue [3]. Previous reports showed that this fungus was most susceptible to the azole class of antifungal agents [4], [5], [6]. Ketoconazole and itraconazole are the most frequently used drugs for the treatment of mycetoma. However, therapy failure is common and high recurrence and amputation rates are reported [7]. Another concern is that both the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) recently restricted the use of ketoconazole due to its toxic side effects (http://www.fda.gov/Drugs/DrugSafety/ucm362415.htm) [8], making the need for an alternative treatment for eumycetoma even more urgent.

Since M. mycetomatis appeared to be most susceptible to the azole class of antifungal agents, a new azole probably has the best chance of meeting that need. A new azole currently under development is ravuconazole. Ravuconazole is a broad-spectrum triazole that showed activity against a wide array of fungal species including Aspergillus spp., Candida spp., and Cryptococcus neoformans [9], [10]. Studies have shown that the efficacy of this new triazole was comparable to that of posaconazole and voriconazole [9], [10], [11]. In addition to antifungal activity, ravuconazole also showed in vitro activity against the parasite Trypanosoma cruzi, the causative agent of Chagas disease, another neglected tropical disease on the WHO list [12]. Eisai developed a prodrug of ravuconazole (E1224) which has a simpler chemical structure, is safe, and has a long half-life in humans [13]. These attributes will reduce the costs of ravuconazole treatment and will make it an affordable drug for people in endemic countries. In the present study, we investigated the antifungal activity of ravuconazole (the active moiety of E1224) against 23 isolates of Madurella mycetomatis.

Materials and Methods

Results

As shown in Table 1, fifty percent of the strains were inhibited by a concentration of 0.063 µg/ml (MIC₅₀) for both ketoconazole and itraconazole, while a concentration of 0.25 µg/ml (MIC₉₀) was required to inhibit 90% of the strains. Significantly lower MICs were obtained with ravuconazole in comparison to ketoconazole and itraconazole (Mann-Whitney, p<0.0001 for both comparisons), with MICs ranging from ≤0.002 to 0.031 µg/ml (Fig. 1). Same results were obtained when using Wilcoxon's signed rank test [Z-value: -4.1973, p-value is 0.00 for both drugs]. Moreover, there is no cross susceptibility among strains showed low MICs for ravuconazole and those of ketoconazole and itraconazole. A concentration of 0.004 µg/ml ravuconazole was needed to inhibit 50% of the strains, whereas 0.016 µg/ml was required to inhibit 90% of them.

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Figure 1. In vitro activities of ketoconazole (KTC), itraconazole (ITC), and ravuconazole (RVC) against 23 isolates of Madurella mycetomatis represented by MICs.

https://doi.org/10.1371/journal.pntd.0002942.g001

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Table 1. In vitro susceptibility of Madurella mycetomatis to ketoconazole, itraconazole, and ravuconazole.

https://doi.org/10.1371/journal.pntd.0002942.t001

Discussion

In this study, we demonstrated that Madurella mycetomatis, the most common etiologic pathogen for mycetoma, was highly susceptible to ravuconazole with MICs ranging from ≤0.002 to 0.031 µg/ml. These MICs were not only considerably lower than those found for ketoconazole and itraconazole in the present study, but they were also lower than those reported for voriconazole (0.016–1 µg/ml), posaconazole (0.03–0.125 µg/ml), and isavuconazole (0.016–0.125 µg/ml) [4], [5], [6]. Only a few reports are available regarding the susceptibility of other eumycetoma causative agents towards ravuconazole. Studies have shown that ravuconazole has inhibitory activity against the black-grain eumycetoma species Exophiala jeanselmei and to the saprobe Curvularia lunata that occasionally has been observed in eumycetoma [10], [16]. In contrast, resistance was reported for the white-grain eumycetoma causative pathogens Pseudallescheria boydii and Fusarium species [10], [16], [17]. Good inhibitory activity of ravuconazole was reported for members of Chaetomium, a genus that was found to be phylogenetically close to the genus Madurella [14], [18]. Low MICs were reported for Chaetomium species ranging from 0.06 to 1 µg/ml, but these values were higher than the results reported in this communication [18]. Studies of the in vitro activity of ravuconazole against the more common pathogenic fungi, including Cryptococcus neoformans, Candida species, Aspergillus species, and the dermatophytes, showed that the drug has activity comparable to that of other triazoles [10], [16], [19], [20]. Moreover, ravuconazole showed potent in vitro activity against the parasite Trypanosoma cruzi [12]. Several studies have been conducted to evaluate the in vivo efficacy of ravuconazole and E1224 using animal models of aspergillosis, candidiasis, and cryptococcosis, with each demonstrating encouraging activity of the drug [21], [22], [23]. In addition, phase 1/2 clinical trials have shown that ravuconazole and E1224 were well tolerated. Ravuconazole had a relatively long half-life of 4–8 days and the peak plasma concentrations of the drug ranged from 1.20 to 6.02 µg/ml when 50–400 mg/day was administrated orally for 14 days [24]. E1224 provides the advantage of more favorable pharmacokinetics with a half-life of ravuconazole (resulting from conversion of E1224 to ravuconazole) of 7.7 to 10.5 days and peak plasma levels of 3.7–379 µg/ml when 200–400 mg/day was administrated orally for 14 days [25]. This serum level of the drug is much higher than the concentration needed to inhibit 90% of the M. mycetomatis strains in the present study (MIC₉₀ of 0.016 µg/ml). Furthermore, in rabbits it was demonstrated that ravuconazole concentrations in the liver, adipose tissue, marrow, kidney, lung, brain and spleen exceeded concurrent plasma concentrations [26]. Moreover, high concentrations were also detected in lung and uterus of rat [27]. Due to these high levels of the drug in tissue, good therapeutic efficacy was obtained in animal models with pulmonary and disseminated aspergillosis, candidiasis, histoplasmosis, intracranial and disseminated cryptococcosis [21], [23], [28], [29]. Based on the in vitro susceptibility generated in this study, the next step will be to study the efficacy of ravuconazole in an animal model of mycetoma.

We conclude that ravuconazole has potent in vitro activity against M. mycetomatis. Compared to other infectious fungi, Madurella is exceptionally susceptible to this drug. With its favorable pharmacokinetic properties and low toxicity, E1224 with its resultant active moiety, ravuconazole, could be a promising antifungal agent for treatment of eumycetoma. A clinical trial is now required for an in vitro-in vivo correlation of the activity of the drug.

Acknowledgments

Ravuconazole was kindly provided by Eisai Co., Ltd. The company had no role in the study design, data collection or data analysis.