Combination of Amphotericin B and Terbinafine against Melanized Fungi Associated with Chromoblastomycosis

ABSTRACT

Our in vitro studies showed that a combination of amphotericin B and terbinafine had synergistic effects against the majority of melanized fungi associated with chromoblastomycosis (CBM) and similar infections, including those with Cladophialophora carrionii, Cladophialophora arxii, Exophiala dermatitidis, Exophiala spinifera, Fonsecaea monophora, Fonsecaea nubica, Fonsecaea pedrosoi, and Phialophora verrucosa. This drug combination could provide an option for the treatment of severe or unresponsive cases of CBM, particularly in cases due to species of Fonsecaea and Cladophialophora.

TEXT

Chromoblastomycosis (CBM) is a serious fungal skin disease associated with significant morbidity (1). The disease is characterized histologically by muriform cells that cause chronic inflammation of the skin and subcutaneous tissues (2, 3). The infection leads to excessive proliferation of host tissue, formation of cauliflower-like eruptions on the skin, or hyperkeratosis, or it may exhibit intermediate forms, depending on the type of interaction between host and fungal cells (4, 5). Because of chronicity, the CBM lesions may also undergo neoplastic transformation leading to skin cancer (6). The chronic nature of the infections seems to be due to inadequate innate recognition and subsequent failure to mount protective inflammatory responses (7).

The disease has worldwide distribution, mainly in tropical and subtropical climates (8). Species in humid climates, particularly members of the genus Fonsecaea (F. pedrosoi, F. monophora, and F. nubica), are prevalent agents of CBM (9). Cladophialophora carrionii is the predominant agent of the disease under arid desert-like climate conditions (10). Sporadic cases of CBM-like infections have also been reported for Cladophialophora arxii (11), Exophiala dermatitidis (12), Exophiala spinifera (13), Phialophora verrucosa (14), and Veronaea botryose (15), although attribution to this disease category has not been confirmed.

CBM is extremely difficult to treat due to its recalcitrant nature, and there is no consensus regarding the treatment of choice (16). Based on open clinical studies and expert opinions, itraconazole is the first-line recommended therapy for CBM (17), followed by terbinafine (18). However, infections by F. pedrosoi strains resistant to itraconazole have been reported (19). Cure rates with itraconazole and terbinafine monotherapy may range from 15 to 80%, which on average, is insufficient (20). When possible, the addition of physical therapeutic methods, such as laser and photodynamic therapy, is recommended (21, 22), which is still associated with rather low cure rates and high refractory rates.

Alternative therapeutic strategies employing newer antifungal agents and/or combination of drugs (23–26) might be promising to treat CBM more efficiently. In a recent study, we also demonstrated that amphotericin B in combination with flucytosine may have a role in the treatment of primary cerebral infections caused by other melanized fungi of the order Chaetothyriales (27). We therefore sought to investigate the in vitro antifungal activity of amphotericin B in combination with terbinafine against a collection of black fungi obtained from patients with CBM.

A collection of 46 isolates of melanized fungi associated with CBM or similar skin infections were studied, including C. carrionii (n = 10), C. arxii (n = 1), Exophiala dermatitidis (n = 9), E. spinifera (n = 3), Fonsecaea monophora (n = 7), F. nubica (n = 5), F. pedrosoi (n = 5), Phialophora verrucosa (n = 3), and Veronaea botryosa (n = 3). The identities of the organisms were confirmed by sequencing of the internal transcribed spacer regions of ribosomal DNA (rDNA), as described previously (28). All isolates were subcultured on malt extract agar (MEA) at 25°C. Conidial suspensions were harvested and suspended in normal saline containing 0.025% Tween 20. Supernatants were adjusted spectrophotometrically at 530-nm wavelengths to optical densities (ODs) that ranged from 0.15 to 0.17 (68 to 71% transmission) for all isolates, except E. dermatitidis, whose ODs ranged from 0.09 to 0.13 (80 to 83% transmission), as described previously (27).

Amphotericin B (Sigma-Aldrich, St. Louis, MO, USA) and terbinafine (Novartis, Arnhem, The Netherlands) were obtained as standard pure powders, and serial dilutions were prepared according to the Clinical and Laboratory Standards Institute (CLSI) broth microdilution guidelines (29). Antifungal susceptibility and drug interaction testing were performed by using the broth microdilution checkerboard (2-dimensional, 8-by-12) method (27). The final concentrations of the antifungal agents ranged from 0.125 to 8 μg/ml for amphotericin B and 0.008 to 8 μg/ml for terbinafine. To assess the nature of in vitro interactions between amphotericin B and terbinafine, the data obtained were analyzed using nonparametric approaches of the following two no (zero)-interaction theories: Loewe additivity, defined as the fractional inhibitory concentration (FIC), and the Bliss independence (BI) parameter, obtained from response surface analysis (30), as described previously (27). Drug interactions were defined as synergistic if the FIC index was <1, additive if the FIC index was 1, and antagonistic if the FIC index was >1 (31). The BI drug interactions were considered synergistic if ΔE was >0 (positive ΔE), indifferent if ΔE was 0, or antagonistic if ΔE was <0 (negative ΔE) (32). All experiments were performed in three independent replicates on different days. All data analyses were performed by using the software package GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). A P value of ≤0.05 was considered significant (two-tailed).

The mean (range) MICs were 4.46 (0.125 to >8) μg/ml for amphotericin B across all isolates and 0.86 (0.16 to >8) for terbinafine (Table 1). For the amphotericin B and terbinafine combinations, the geometric mean FIC indices, in increasing order, were 0.41 for F. monophora (∑FIC range, 0.25 to 0.5), 0.5 for E. spinifera (∑FIC range, 0.25 to 1), 0.63 for E. dermatitidis (∑FIC range, 0.25 to 1), 0.7 for C. carrionii (∑FIC range, 0.5 to 1), 0.72 for P. verrucosa (∑FIC range, 0.5 to 1), 0.76 for F. nubica (∑FIC range, 0.25 to 1), 0.76 for F. pedrosoi (∑FIC range, 0.5 to 1), and 1 for C. arxii (n = 1), which indicate synergy and additivity for these strains. However, antagonism was noted in V. botryosa isolates, with a mean FIC value of 1.4 (∑FIC range, 1 to 2).

TABLE 1.

MIC, FIC indices, and Bliss independence results for the in vitro combination of AmB and TBF against melanized fungi associated with chromoblastomycosis

Strain no.	Fungal species	Strain	Source	Origin	MIC (μg/ml)a		FIC index	Bliss independence index
					TBF	AmB
1	Cladophialophora carrionii	CBS 131844	Human, chromoblastomycosis	China	0.031	1	0.75	111.3
2		CBS 131854	Human, chromoblastomycosis	Madagascar	0.063	8	0.5	69.24
3		CBS 131833	Human, chromoblastomycosis	China	1	8	1	−4.48
4		CBS 131847	Human, chromoblastomycosis	China	0.031	4	0.5	410.73
5		CBS 160.54	Human, chromoblastomycosis	Australia	0.031	1	0.75	−8.29
6		CBS 859.96	Dry plant debris	Venezuela, arid zone west of Coro	0.016	1	0.75	−40.42
7		CBS 863.96	Dry plant debris	Venezuela, arid zone west of Coro	0.016	2	0.75	6.37
8		CBS 131736	Soil	Venezuela, arid zone west of Coro	0.031	4	0.5	75.15
9		CBS 860.96	Dry plant debris	Venezuela, arid zone west of Coro	0.016	4	0.75	893.89
10		CBS 861.96	Dry plant debris	Venezuela, arid zone west of Coro	0.125	8	1	143.51
11	Cladophialophora arxii	CBS 102461	Human, brain abscess	USA	0.5	4	1	90.2
12	Exophiala dermatitidis	CBS 120542	Human or animal, stool	Slovenia	0.5	4	0.5	138.26
13		CBS 120562	Human, keratitis	USA	0.5	0.25	1	151.7
14		CBS 120473	Human, brain	USA	0.25	0.5	1	−4,736
15		CBS 424.67	Human, chromoblastomycosis	Germany	0.5	0.125	1	−11.78
16		CBS 550.9	Human, sputum, cystic fibrosis	Germany	0.031	2	1	258.1
17		CBS 126590	Human, sputum, cystic fibrosis	The Netherlands	0.5	1	0.25	89.2
18		CBS 120550	Steam bath	Austria	0.5	2	0.5	133.05
19		CBS 120483	Flying fox's feces	Thailand	0.25	4	0.25	346.03
20		CBS 109138	Hall of sauna complex	The Netherlands	0.5	4	1	−43.35
21	Exophiala spinifera	CBS 899.68	Human, nasal granuloma	USA	2	2	1	62.94
22		CBS 269.28	Human, chromoblastomycosis	Unknown	0.5	8	0.5	64.9
23		CBS 194.61	Human, systemic mycosis	India	0.5	>8	0.25	450.11
24	Fonsecaea monophora	CBS 117236	Human, brain abscess	USA	0.5	8	0.5	305
25		CBS 269.37	Unknown, chromoblastomycosis	Unknown	0.25	8	0.5	209.7
26		CBS 117238	Unknown, brain	England	5	8	0.25	49.7
27		CBS 122742	Human, chromoblastomycosis	China	0.5	8	0.5	14.98
28		CBS 100430	Human, brain	Africa	0.5	8	0.5	311.02
29		CBS 102229	Decaying vegetable	Piraquara, Paraná, Brazil	0.5	4	0.5	27.6
30		CBS 289.93	Animal, lymph node, aspiration biopsy	The Netherlands	8	8	0.25	526.72
31	Fonsecaea nubica	CBS 277.29	Human, chromoblastomycosis	Brazil	1	4	1	154.1
32		CBS 444.62	Human, chromoblastomycosis	Suriname	0.5	8	1	−7.07
33		CBS 122733	Human, chromoblastomycosis	China	0.25	4	1	−69.4
34		CBS 269.64	Human, chromoblastomycosis	Cameroon	0.5	8	0.75	−79.6
35		CBS 125198	Human, chromoblastomycosis	China	0.25	8	0.25	−223.17
36		CBS 127264	Human, chromoblastomycosis	Mexico	1	4	1	−271.7
37	Fonsecaea pedrosoi	CBS 102247	Human, chromoblastomycosis	Paraná, Brazil	0.5	4	0.5	123.76
38		CBS 285.47	Human, chromoblastomycosis	Puerto Rico	0.5	4	0.5	298.9
39		CBS 122739	Human, chromoblastomycosis	Mexico	0.5	4	1	114.3
40		CBS 117910	Human, chromoblastomycosis	Venezuela	0.5	4	1	297.27
41		CBS 671.66	Soil	Venezuela	0.5	2	1	64.1
42	Phialophora verrucosa	CBS 120349	Plant	China	0.5	4	1	123.76
43		CBS 262.93	Exudate from right hand (human or animal)	Germany	0.016	0.5	0.5	52941
44		CBS 115.89	Disseminated (human or animal)	Libya	0.25	8	0.75	−47.99
45	Veronaea botryosa	CBS 122826	Railway tie treated with creosote for 20 years	Brazil	1	4	2	−271.7
46		CBS 121506	Cutaneous lesion, wrist	Japan	>8	2	1	160.1

^aThe final concentration range for TBF was 0.008 to 8 μg/ml, and that for AmB was 0.125 to 8 μg/ml.

The Bliss independence drug interaction analysis for the amphotericin B and terbinafine combination resulted in a synergistic interaction for 71.74% (33/46) of the strains tested. The degree of synergy was the highest among the C. carrionii strains (sum ΔE, 1,546%), followed by F. monophora (sum ΔE, 1,140%), F. pedrosoi (sum ΔE, 775%), E. spinifera (sum ΔE, 515%), P. verrucosa (sum ΔE, 481%), E. dermatitidis (sum ΔE, 449%), and C. arxii (sum ΔE, 90%). The strongest synergistic interactions were found at amphotericin B and terbinafine concentration ranges of 0.125 to 0.5 μg/ml and 0.008 to 0.5 μg/ml, respectively. Examples of Bliss independence 3-dimensional plots for the synergistic and antagonistic interactions of amphotericin B and flucytosine are shown in Fig. 1.

FIG 1.

Interaction surfaces obtained from response surface analysis of Bliss independence no-interaction model for in vitro combination of amphotericin B (AmB) plus terbinafine (TBF). The x and y axis represent the efficacy of AmB and TBF, respectively. The z axis is the ΔE (%). The 0-plane represents Bliss independent interactions, whereas the volumes above the 0-plane represent statistically significantly synergistic (positive ΔE) interactions. The magnitude of interactions is directly related to ΔE. The different tones in three dimensional plots represent different percentile bands of synergy. The highest level of synergistic interactions was found between 0.25 μg/ml amphotericin B and terbinafine concentrations in the range of 0.008 to 0.5 μg/ml. (A) Synergistic interaction of AmB plus TBF against an Exophiala spinifera strain (CBS 194.61) (AmB MIC, 0.5 μg/ml; TBF MIC, >8 μg/ml). The mean ΔE ± standard error of the mean and sum ΔE were 5.36% ± 1.81% and 450.11%, respectively. The highest level of synergistic interactions was found between 0.25 μg/ml amphotericin B and terbinafine concentrations in the range of 0.008 to 0.5 μg/ml. (B) Antagonistic interaction of AmB plus TBF against a Veronaea botryosa strain (CBS 122826) (AmB MIC, 1 μg/ml; TBF MIC, 4 μg/ml). The mean ΔE ± standard error of the mean and sum ΔE were −3.23% ± 1.70% and −271.70%, respectively.

Overall, our results show that the amphotericin B and terbinafine combination has synergistic effects against the majority of melanized fungi associated with CBM, including C. carrionii, C. arxii, E. dermatitidis, E. spinifera, F. monophora, F. nubica, F. pedrosoi, and P. verrucosa. The results of FIC analysis were supported by response surface analysis using a Bliss independence no-interaction model for the isolates tested.

Terbinafine is one of most commonly used antifungal agents in the treatment of patients with CBM (18), due to its high degree of effectiveness and tolerability. In an athymic murine model of CBM caused by F. pedrosoi, terbinafine, especially at the highest dose, was able to reduce the inflammatory response to the infection to levels similar to those with azoles (33), although a total cure in patients with CBM remains difficult to achieve (26, 34). On the other hand, various formulations of amphotericin B have been developed and are now available in most countries (35). The compound is nevertheless not recommended as a first-line therapy in chronic infections because of its adverse effects, such as nephrotoxicity, neurotoxicity, hematological side effects, and allergic reactions (36). However, the use of combination therapy can reduce cost- and toxicity-related effects and may prevent the emergence of resistance (35). Combination therapy is also recommended in salvage therapy scenarios for patients with antifungal-resistant and invasive refractory mycoses (37). Few studies have reported data on the efficacy of antifungal combination therapy in the treatment of severe and refractory CBM. Treatment with amphotericin B and a subsequent combination of flucytosine and itraconazole was shown to be effective in a patient with a CBM-like infection caused by P. verrucosa (23). Combinations of itraconazole with flucytosine (24, 25) and itraconazole with terbinafine have also shown better efficacy than monotherapy for CBM caused by F. pedrosoi (26) and F. monophora (38). In general, however, combination therapy still is inadequate, requiring long-term therapy at high doses, and treatment failure of CBM remains common. The in vitro results obtained in the present study confirmed that terbinafine is active against the majority of strains tested. Of the nine species investigated, Cladophialophora carrionii and Phialophora verrucosa were more sensitive to terbinafine than species of Fonsecaea and Exophiala. The three species of Fonsecaea showed similar degrees of susceptibility. As in previous reports (39–41), in our study, E. spinifera and V. botryosa were resistant to terbinafine and amphotericin B when used alone. Although a synergistic interaction was found in a combination setting for E. spinifera, the combination of terbinafine and amphotericin B exhibited an indifferent interaction for tested isolates of Veronaea botryosa. In the current study, a wide range of amphotericin B MICs (0.125 to >8 μg/ml) was observed for agents of CBM. Exophiala dermatitidis and P. verrucosa were the species being relatively susceptible, which is in agreement with previous studies (27, 42). When terbinafine and amphotericin B were used in combination, the highest synergy was shown for F. monophora and E. spinifera, followed by E. dermatitidis, C. carrionii, F. nubica, and F. pedrosoi. Our findings agree with those of Daboit et al. (43), demonstrating in vitro synergy between amphotericin B and terbinafine for Fonsecaea spp., C. carrionii, and P. verrucosa. Biancalana et al. (44) also reported 96.5% in vitro synergy between terbinafine and amphotericin B against clinical isolates obtained from cases of phaeohyphomycosis and CBM, including F. pedrosoi, Curvularia spp., Exophiala jeanselmei, Alternaria alternata, Cladophialophora bantiana, and Bipolaris species. In contrast, Yu et al. (45) did not find an interaction for this combination against agents for CBM.

Overall, the management of CBM is complicated and requires long-term antifungal therapy, surgery, thermotherapy, chemotherapy, or combinations of these (3). Importantly, the clinical experience with posaconazole and voriconazole is limited for CBM. However, the good in vitro activities and in vivo efficacies of these agents against dematiaceous fungi (46–48), together with the tolerance of the drug in long-term therapies, suggest that further studies are warranted to evaluate the potential use of these drugs for the treatment of CBM.

Collectively, the present study demonstrated that the combination with terbinafine allows a significant reduction in amphotericin B MICs and could be an option for severe or unresponsive cases of CBM, particularly in cases due to Fonsecaea and Cladophialophora species, and in E. spinifera. Our results therefore suggest that a combination of amphotericin B and terbinafine may have a promising role in the treatment of CBM.