Drug Combinations against Mucor irregularis In Vitro

Abstract

Combinations of terbinafine or caspofungin with amphotericin B, posaconazole, or itraconazole were studied as potential treatments against 18 isolates of Mucor irregularis in vitro. Synergism of the combinations of terbinafine with amphotericin B, posaconazole, and itraconazole against 38.9, 33.3, and 44.4% of the strains studied was observed. In contrast, synergism of the combinations of caspofungin with amphotericin B, posaconazole, and itraconazole against 99.4, 66.7, and 99.4% of the strains studied was observed. Furthermore, no antagonism was observed.

TEXT

Cutanous zygomycosis is a fungal infection caused mainly by members of the order Mucorales that affect the skin. The most frequently reported genus in the English literature is Rhizopus, followed by Lichtheimia spp. and Rhizomucor pusillus (1). Cutaneous zygomycosis caused by Mucor irregularis (formerly Rhizomucor variabilis) is an emerging disease in China that is characterized by progressive swelling, ulceration, and destruction of involved skin at exposed sites, especially the central face, and leads to severe disfigurement and even death if left untreated (2–4). Notably, it seemed that most of the patients infected with M. irregularis were immunocompetent (2–9).

Amphotericin B is the most potent drug used for the treatment of M. irregularis infection, but the toxicity of amphotericin B limits its long-term clinical application. Posaconazole has been used primarily as salvage therapy, but at present, there is no strong published clinical evidence supporting its role as a single agent for the treatment of mucormycosis (10). Itraconazole has variable effectiveness against M. irregularis in vitro (11) but was proven to be effective against some M. irregularis infections (2, 5). Terbinafine is an allylamine antifungal agent used primarily to treat dermotropic infections and onychomycosis but has potential for use in adjunct therapy in combination with azoles, polyenes, or echinocandins in the management of severe drug-resistant or refractory mycosis (12). Besides, terbinafine could reach high concentrations in the stratum corneum and sebum because of its lipophilic nature (13), which provided the possibility that terbinafine can be used in combination therapies to treat cutaneous zygomycosis.

To our knowledge, with the exception of some reported cases (7, 9), no susceptibility of M. irregularis to combinations of antifungal drugs in vitro has been reported. In the present study, the combinations of terbinafine or caspofungin with amphotericin B, posaconazole, or itraconazole were assessed against M. irregularis isolates in vitro.

A total of 18 M. irregularis patient isolates preserved by the Research Center for Medical Mycology at Peking University were tested. Isolates were identified by both conventional morphological methods and the internal transcribed spacer sequence analysis method (11).

Drug combinations were conducted by using a checkerboard method based on Clinical and Laboratory Standards Institute document M38-A2 (14) that provided the MICs of each agent alone on the same plate, as previously described (15). Candida parapsilosis ATCC 22019 and Aspergillus flavus ATCC 204304 were used as quality controls. The final drug concentrations were 0.0625 to 4 μg/ml for amphotericin B (Sigma-Aldrich), 0.125 to 8 μg/ml for posaconazole (Merck Sharp & Dohme, Rahway, NJ), 0.25 to 16 μg/ml for itraconazole (Shouguang Fukang), 0.25 to 128 μg/ml for caspofungin (Merck Australia), and 0.5 to 256 μg/ml for terbinafine (Novartis), depending on the susceptibility of M. irregularis to these drugs.

The isolates tested grew for 5 to 7 days on potato dextrose agar at 30°C. Sporangiospore suspensions were counted with a hemocytometer and then suspended in RPMI 1640 medium to a concentration of 4 × 10⁴ spores/ml (twice the final concentration). MICs were determined visually with a concave mirror after incubation at 35°C for 24 h. The interaction of each pair of agents was evaluated by the fractional inhibitory concentration index (FICI), where an FICI of ≤0.5 shows synergy, an FICI of 0.5 to 4 shows no interaction, and an FICI of >4 shows antagonism. All experiments were performed in duplicate.

The in vitro susceptibilities of the 18 M. irregularis isolates tested to the five drugs alone are shown in Table 1. Amphotericin B was the most potent agent against M. irregularis (MIC range, 0.5 to 2 μg/ml; geometric mean [GM] MIC, 0.54 μg/ml); only one isolate produced a high MIC of 2 μg/ml. Posaconazole was the second most potent agent (MIC range, 1 to 4 μg/ml; GM MIC, 1.92 μg/ml). The MICs of itraconazole varied from 1 to >16 μg/ml. Terbinafine and caspofungin were highly inactive against M. irregularis.

Table 1.

Susceptibilities of tested drugs alone against 18 M. irregularis isolates

Druga	MIC range	GM MICb	MIC90	MIC50
AMB	0.5–2b	0.54	0.5	0.5
POS	1–4	1.92	4	2
ITC	1–>16	4.16	>16	4
TRB	128–256	237	256	256
CAS	128–>128	>128	>128	>128

^aAMB, amphotericin B; POS, posaconazole; ITC, itraconazole; TRB, terbinafine; CAS, caspofungin.

^bValues are in micrograms per milliliter.

MIC range, FICI, and synergism rate data for the drug combinations are displayed in Table 2. A synergistic effect was obtained with all of the drug combinations used. Caspofungin had better synergistic efficacy with amphotericin B, posaconazole, or itraconazole than did terbinafine, as shown in Table 2 (Pearson χ² test, P < 0.05). The highest synergism rates were obtained with the combinations of amphotericin B plus caspofungin and itraconazole plus caspofungin.

Table 2.

MICs of drug combinations, FICI ranges, and ratios of strains for which the drug combinations showed synergy

Antifungal combinationa	MICs in μg/ml (range)	FICIb range	Ratio (%)c
TRB-AMB	4–128 (0.0625–0.25)	0.09–1	38.9
TRB-POS	4–128 (0.125–2)	0.16–0.75	33.3
TRB-ITC	4–64 (0.5–2)	0.14–1.02	44.4
CAS-AMB	2–8 (0.0625–1)	0.14–0.56	94.4
CAS-POS	2–32 (0.25–1)	0.25–0.63	66.7
CAS-ITC	4–32 (0.25–2)	0.19–0.53	94.4

^aAMB, amphotericin B; TRB, terbinafine; CAS, caspofungin; POS, posaconazole; ITC, itraconazole.

^bFICI of ≤0.5, synergy; FICI of 0.5 to 4, no interaction; FICI of >4, antagonism.

^cPercentage of strains indicating synergy of drug combinations.

In all of the combinations showing synergism, significantly lower MICs were achieved with amphotericin B (0.0625 to 0.125 μg/ml), posaconazole (0.125 to 1 μg/ml), and itraconazole (0.25 to 2 μg/ml). The GM MICs decreased, on average, 18-fold for terbinafine and 36-fold for caspofungin. It should be noted that the MICs of terbinafine (4 to 64 μg/ml) and caspofungin (2 to 32 μg/ml) were still high. Antagonism was absent from our study.

Previous studies have shown synergistic interactions between terbinafine and amphotericin B (16, 17), as well as the combination of terbinafine and itraconazole (16), against other zygomycete species. In fact, synergistic interactions between terbinafine and polyenes or azoles against other species, like Fonsecaea pedrosoi, Exophiala jeanselmei, Scopulariopsis brevicaulis, Scedosporium prolificans, and Aspergillus spp., have also been found (18–21).

An explanation for the synergism between terbinafine and amphotericin B, posaconazole, or itraconazole may be that they inhibit or disrupt the ergosterol in the fungal membrane through different pathways or different enzymes.

Caspofungin has no activity against zygomycetes, for it influences the synthesis of β-1,3-glucan, a component that is lacking in zygomycetes. However, we observed a high rate of synergism when the drug was combined with amphotericin B, posaconazole, or itraconazole. Notably, synergism with caspofungin was easier to acquire than synergism between terbinafine and amphotericin B, posaconazole, or itraconazole in our study. Guembe et al. (22) observed 100% synergism against 12 zygomycetes with the combination of posaconazole and caspofungin. Drogari-Apiranthitou et al. (23) studied potential interactions between anidulafungin and amphotericin B against 21 mucormycetes and observed synergism against one Mucor circinelloides isolate and no antagonism. Further, synergism of caspofungin-amphotericin B and caspofungin-posaconazole combinations against M. irregularis isolates has been observed, according to a few case reports (7, 8). Besides, previous research has shown that an echinocandin plus a polyene could markedly improve the survival of mice with diabetic ketoacidosis and disseminated mucormycosis (24, 25). Moreover, a combination of caspofungin with liposomal amphotericin B was used to treat a rhinocerebral zygomycosis case successfully (26).

The mechanism of the synergism of the combination of caspofungin with amphotericin B, posaconazole, or itraconazole against zygomycetes could be interpreted as follows. Some zygomycetes do express the FKS1 gene (27), which is a target of echinocandins, so that maybe a change in the membrane environment caused by other drugs could make FKS1 more accessible to echinocandins (22, 23).

In conclusion, only synergism or indifference was obtained with the drug combinations described above. However, the clinical relevance of these findings is unknown. Further animal models and clinical data are required to verify these interactions.