ABSTRACT ORIGINAL ARTICLE

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1 Microbiol Immunol 2014; 58: 1 8 doi: / ORIGINAL ARTICLE In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes Takashi Tamura 1,2, Miwa Asahara 1, Mikachi Yamamoto 3, Mariko Yamaura 3, Mitsuru Matsumura 1, Kazuo Goto 1, Ali Rezaei Matehkolaei 4, Hossein Mirhendi 5, Miho Makimura 6 and Koichi Makimura 1,3,6,7,8 1 Department of Clinical Laboratory Science, Graduate School of Medical Technology, 2 Eiken Chemical, Taito, Taito ku, Tokyo , Japan, 3 Laboratory of Space and Environmental Medicine, Graduate School of Medicine, 4 Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Golestan Avenue, Ahvaz, 5 Department of Medical Parasitology and Mycology, School of Public Health and National Institute of Health Research, Tehran University of Medical Sciences, Tehran , Iran, 6 Teikyo University Institute of Medical Mycology, 7 Department of Biomedicine, General Medical Education Center and 8 Asia International Institute of Infectious Disease Control, Teikyo University, , Kaga, Itabashi, Tokyo ABSTRACT To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis causing non dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis. Key words antifungal combination, fractional inhibitory concentration index, Trichophyton mentagrophytes, Trichophyton rubrum. Correspondence Koich Makimura, Laboratory of Space and Environmental Medicine, Graduate School of Medicine, Teikyo University, Kaga, Itabashi ku, Tokyo , Japan. Tel: þ ; fax: þ ; makimura@med.teikyo-u.ac.jp Received 2 August 2013; revised 25 October 2013; accepted 28 October List of Abbreviations: CLSI, Clinical Laboratory Standard Institute; FIC, fractional inhibitory concentration; MIC, minimum inhibition concentration The Societies and Wiley Publishing Asia Pty Ltd 1

2 T. Tamura et al. A group of fungi that infect keratinized tissues (skin, hair, and nails) of humans and animals cause dermatomycoses, including tinea. The major dermatophytes that cause tinea are Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum. In addition, Candida spp. and non dermatophytic molds have also been reported as causes of dermatomycosis (1). Several antifungal agents have been developed and used for internal and/or external treatment of dermatomycoses. Azole antifungal agents, such as ketoconazole, itraconazole and bifonazole, inhibit lanosterol 14a demethylase and block fungal membrane ergosterol biosynthesis in the cell (1, 2). The non azole antifungal agent, amorolfine, blocks other pathways of D14 sterol reductase and D7 D8 steroid isomerase in fungal cells (3). Terbinafine, an allylamine, inhibits fungal squalene epoxidase (1). Itraconazole and terbinafine have been approved in the USA and amorolfine and fluconazole have been approved in Europe for treatment of onychomycoses (2). Onychomycoses are often recurrent, chronic, and generally require long term treatment with antifungal agents (4). It is desirable to choose appropriate antifungal drugs in the early stages of infection. In addition, it is practical to consider appropriate combinations of internal and external antifungal drugs with different pharmacological effects to treat refractory fungal infection, especially onychomycosis. There have been many previous studies of double or triple drug combination therapy (3 17). These reports suggest the usefulness of combinations of external and internal antifungal agents; however, there have been few reports presenting quantitative data regarding drug combinations in vitro (6, 7, 9). Here, we investigated the susceptibility of major dermatophytes and non dermatophytic fungi responsible for superficial fungal infection to six antifungal agents: amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole. We also investigated the synergistic or additive effect of an antifungal combination. We choose two antifungals in common use, amorolfine and itraconazole, which have different mechanisms of actions and administration routes (amorolfine is an external agent for topical use and itraconazole an internal agent for systemic use). We used the FIC index to quantify the efficacy of a combination of amorolfine and itraconazole in 27 strains of dermatophytes. MATERIALS AND METHODS Strains The strains investigated in this study are shown in Table 1 (Cl I and Sz k were clinical isolates). One standard strain (TIMM2789, T. mentagrophytes (Arthroderma vanbreuseghemii)) and 43 clinical isolates of major pathogenic dermatophytes were used; namely, 14 strains of T. rubrum, 14 strains of T. mentagrophytes human type (18) (synonym, Trichophyton interdigitale (anthropophilic)) (19), three strains of Trichophyton tonsurans, one strain of T. verrucosum, two strains of M. canis,four strains of M. gypseum and five strains of E. floccosum. In addition, 10 strains of non dermatophytes were also used; namely, two strains of Aspergillus fumigatus, two strains of Geotrichum candidum, two strains of Scopulariopsis brevicaulis, two strains of Fusarium oxysporum, one strain of Fusarium verticillioides and one strain of Fusarium solani. All isolates were identified using a molecular basedmethodreportedpreviously(18 21). Preparation of isolates The test isolates were subcultured onto 1/10 Sabouraud dextrose agar (peptone, 1 g; glucose, 4 g; distilled water, 1 L; agar, 15 g; ph 6.0) plates and incubated at 30 C for 7 days. Some poor growth strains were cultivated for extended times of up to 14 days. Antifungal agents The following six antifungal agents were assessed: amorolfine, terbinafine, butenafine, ketoconazole, itraconazole, and bifonazole (Wako Pure Chemical Industries, Osaka, Japan). The antifungal drugs were dissolved in dimethylsulfoxide. According to CLSI protocol M38 A2 (22), serial twofold dilutions were prepared with powdered RPMI 1640 medium (Gibco, Grand Island, NY, USA) and buffered with 3 (N morpholino)propanesulfonic acid at ph 7.0. to reach final concentrations of mg/ml for amorolfine, mg/ml for terbinafine, mg/ml for butenafine, mg/ml for ketoconazole, mg/ml for itraconazole and mg/ml for bifonazole with RPMI 1640 test medium. To calculate the FIC index, a checkerboard was designed with amorolfine ( mg/ml) and itraconazole ( mg/ml). Inoculum preparation The subcultured isolates were collected with sterile swabs and suspended in 2 ml of sterile 0.85% saline. Conidia suspensions were filtered with sterile gauze and the concentrations quantified with a hemocytometer to adjust to McFarland No. 1 (10 6 conidia/ml). Minimum inhibitory concentration tests Antifungal susceptibility tests were performed by a broth microdilution method according to modified CLSI protocol M38 A2 (22). Briefly, aliquots of 100 ml ofeach The Societies and Wiley Publishing Asia Pty Ltd

3 Antifungal combination Table 1. Strains assessed Dermatophytes Non dermatophytes Species Strains Species Strains Trichophyton rubrum Cl I 488 Aspergillus fumigatus ATCC Cl I 651 TIMM 3968 Cl I 705 Geotrichum candidum TIMM 0963 Cl I 713 TIMM 0697 Cl I 714 Scopulariopsis brevicaulis TSY 0668 Cl I 724 NBRC 4843 Cl I 725 Fusarium oxysporum TSY 0351 Cl I 726 NBCB Cl I 729 Fusarium solani TSY Cl I 732 Fusarium verticillioides TSY 0219 Cl I 733 Cl I 760 Cl I 772 Cl I 775 Trichophyton mentagrophytes Cl I 90 Cl I 558 Cl I 703 Cl I 706 Cl I 709 Cl I 710 Cl I 723 Cl I 728 Cl I 743 Cl I 744 Cl I 756 Cl I 757 Cl I 763 Cl I 764 TIMM2789 Trichophyton tonsurans Cl I 718 Cl I 747 Cl I 762 Trichophyton verrucosum Cl I 749 Microsporum canis Cl I 755 Cl I 770 Microsporum gypseum Cl I 643 Cl I 727 Sz k 1 Sz k 2 Epidermophyton floccosum Cl I 701 Cl I 711 Cl I 740 Cl I 745 Cl I 746 antifungal agent was poured into the wells of 96 well microplates and stored at 70 C until use. Conidia suspension was diluted tenfold with sterile saline and 2 ml inoculatedinto100ml of RPMI1640 test medium. The microplates were incubated at 30 C for 3 7daysuntil the drug free control well was fully occupied by fungal growth. The MIC was defined as the minimal concentration required to inhibit 80% of the growth in the drug free control well, this assessment being made on a visual basis (22 29). Cumulative percentage curves of dermatophytes Cumulative MIC percentage curves were used to permit visual analysis of MIC distribution (30). Cumulative 2013 The Societies and Wiley Publishing Asia Pty Ltd 3

4 T. Tamura et al. Table 2. MIC ranges of dermatophytes MIC ranges of antifungal agents Strains (number of isolates) Amorolfine Terbinafine Butenafine Ketoconazole Itraconazole Bifonazole T. rubrum (n ¼ 14) T. mentagrophytes (n ¼ 16) T. tonsurans (n ¼ 3) T. verrucosum (n ¼ 1) M. canis (n ¼ 2) M. gypseum (n ¼ 4) E. floccosum (n ¼ 5) percentage curves of six antifungal agents for T. rubrum, T. mentagrophytes and 44 strains of clinically isolated dermatophytes were calculated. Calculation and evaluation of FIC index Reading and interpretation of the results of combination examinations were performed in accordance with the method of Santos et al. (9). The interactions between antifungal agents (drugs A and B) were quantitatively evaluated by the FIC index, which was calculated according to the formula (MIC of A in combination/ MIC of A) þ (MIC of B in combination/mic of B). The interaction was defined as synergistic if the FIC index was 0.5, additive if it was >0.5 but 1, no interaction if it was 2 and antagonistic if it was >2. RESULTS Minimum inhibitory concentrations of antifungal agents in isolates All isolates grew in 1/10 Sabouraud agar after 3 14 days of incubation. Aspergillus spp. and Fusarium spp. Grew relatively quickly (about 3 days) and T. rubrum and Microsorum spp. relatively slowly (7 14 days). After genomic identification, each isolate was subjected to MIC assay. The MIC values of the six assessed antifungal agents for dermatophytes are listed in Table 2 and those for non dermatophytes in Table 3. The six antifungal agents inhibited growth of dermatophytes, but showed markedly higher and wider MIC distribution in nondermatophytes. In particular, Fusarium spp. were insensitive to all anti dermatophytic agents assessed in this study. The cumulative MIC percentage curves of the six antifungal agents for dermatophytes are shown in Figure 1. For two major causes of dermatomycoses, T. rubrum and T. mentagrophytes, MIC ranges of non azole agents were narrower than those of azole agents. The MICs of total dermatophytes showed the same tendency (solid line). Unexpectedly, there were marked differences between T. rubrum and T. mentagrophytes in the MIC ranges of ketoconazole and bifonazole. Results for the combination of amorolfine and itraconazole Table 4 presents a summary of the FIC indexes of 27 clinical dermatophyte isolates. Synergistic interactions were observed in 7 of 27 strains with FIC indexes of 0.5, additive interactions in 16 isolates with FIC indexes >0.5 1 and four isolates had FIC indexes of 2 (no interaction). In total, the combination of amorolfine and itraconazole had synergistic or additive effects in 23 Table 3. MIC ranges of non dermatophytes Species Strains Amorolfine Terbinafine Butenafine Ketoconazole Itraconazole Bifonazole Geotrichum candidum TIMM > >64 TIMM > >64 Scopulariopsis brevicaulis TSY >8 2 NBRC >0.5 2 >8 4 Aspergillus fumigatus ATCC >64 >0.5 > TIMM 3968 >64 >0.5 > Fusarium oxysporum TSY 0351 >64 >0.5 >0.5 >8 >8 >64 NBCB >64 >0.5 >0.5 >8 >8 >64 Fusarium solani TSY >0.5 >0.5 >8 >8 >64 Fusarium verticillioides TSY 0219 >64 >0.5 > > The Societies and Wiley Publishing Asia Pty Ltd

5 Antifungal combination Fig. 1. Cumulative percentage curves of T. rubrum (n ¼ 14) and T. mentagrophytes (n ¼ 15) and 44 clinically isolated dermatophytes showing a clear difference between them in the distribution of MIC. clinical isolates (85%), and no antagonistic effects were detected. DISCUSSION In the present study, we observed differences between T. rubrum and T. mentagrophytes in the MIC ranges of azole agents (ketoconazole and bifonazole), T. rubrum being more sensitive than T. mentagrophytes to these azoles (Fig. 1). Previously, Barros et al. reported that there were no significant differences between T. rubrum and T. mentagrophytes in the efficacies of any of the drugs they tested (fluconazole, itraconazole, griseofulvin and terbinafine) (26). Santos et al. also reported no significant differences between MIC values of various antifungals (fluconazole, itraconazole, griseofulvin, terbinafine, ketoconazole and cyclopiroxamine) in T. rubrum and T. mentagrophytes (9).That our results do not match those previously reported indicates that antifungal susceptibility may differ among populations; further studies of MIC values are therefore required even in these major dermatophytes. The MIC ranges of the non azole agents amorolfine, terbinafine and butenafine against Trichophyton spp. were relatively narrow compared to those of azole agents (Fig. 1; Table 2). One possible explanation for this finding concerns the mechanisms of these drugs. Each azole inhibits one pathway of the ergosterol constructional system, whereas the morpholine agents act on two enzymes involved in ergosterol construction (3). Because the probability that variations in two enzymes will occur simultaneously is low, different positions of action may result in non azoles such as amorolfine having more stable antifungal effects than azoles The Societies and Wiley Publishing Asia Pty Ltd 5

6 T. Tamura et al. Table 4. FIC indexes of dermatophytes Species Strains FIC index Evaluation T. rubrum Cl I Synergistic Cl I Cl I Additive Cl I Cl I Cl I Cl I Cl I Cl I Cl I No interaction Cl I T. mentagrophytes Cl I Synergistic Cl I Cl I Cl I Cl I Additive Cl I Cl I Cl I T. tonsurans Cl I Synergistic T. verrucosum Cl I Additive M. gypseum Sz k Additive Cl I Sz k E. floccosum Cl I Additive Cl I No interaction Cl I FIC index 0.5 is synergistic, >0.5 to 1 additive, 2 no interaction and >2 antagonistic. Minimum inhibitory concentrations varied widely among non dermatophyte strains (Table 3). In particular, all antifungal agents showed high MICs in Fusarium spp. The variation of susceptibility seen in dermatophytic and non dermatophytic fungi indicates the necessity to identify the causative fungi to enable appropriate selection of effective antifungal drugs in each case and to avoid development of resistance (31 33). Several strategies using single or plural antifungals have been reported for treating refractory dermatomycoses, (3 17). Amorolfine is effective in several dermatophytoses, especially tinea unguium (1, 3, 5, 6); however, it is only used topically. For systemic use, itraconazole or terbinafine is generally available. Lecha et al. (3) and Baran et al. (5) described satisfactory results using combinations of amorolfine and terbinafine or itraconazole, respectively, in vivo. We selected amorolfine and itraconazole to investigate combinations of antifungal drugs. The former is a nonazole agent that is used topically (externally) and the latter an azole drug that is used systemically (internally). Both agents are commonly used for dermatomycoses. We observed a synergistic effect in 7 of 27 strains with FIC indexes 0.5. Using a checkerboard method, Santos et al. demonstrated synergistic interactions between azoles and cyclopiroxamine against T. rubrum and T. mentagrophytes (9). Harman et al. alsoreportedasynergisticeffect(1) of a combination of amorolfine and itraconazole in 46% of all organisms tested, including dermatophytes and nondermatophytes(6).inthepresentstudy,weusedastricter criterion for determination of synergy (0.5) and confirmed that a combination of these drugs had a synergistic (0.5) effect in 25.9% of samples and an additive (FIC index 1 and 0.5) effect in 59.3% of samples. In total, these agents showed additive or synergistic effects on more than 85% of the strains examined. In particular, we found additive or synergistic effects in 19 of 21 Trichophyton strains (90%) and in three strains of M. gypseum (100%). We identified no additive or synergistic effects in twoof three strains of E. floccosum and detected no antagonistic effects in any of the 27 dermatophytes. These results suggest that the combination of these two drugs can be expected to act additively or synergistically in the treatment of dermatomycoses. Further investigation is required to examine the effects of antifungal drug combination against these and other clinically important dermatophytes. Although several studies have examined the synergic effects of antifungal agents (34, 35), few have provided explanations for the mechanisms of drug synergy (36). In this study, we found additive or synergistic effects of amorolfine and itraconazole in most of dermatophytes; we do not have an explanation for this. To ascertain the mechanisms of drug synergy between amorolfine and itraconazole, we need to profile changes in cellular environment after drug administration. ACKNOWLEDGMENTS The authors thank the participating laboratories and hospitals for their cooperation and for providing the fungal isolates described in this report. DISCLOSURE K.M. has received research grants from the following companies: Hisamitsu Pharmaceutical (Tokyo, Japan), Seikagaku Biobusiness (Tokyo, Japan), Kaken Pharmaceutical (Tokyo, Japan), Dai Nippon Sumitomo Pharmaceutical (Tokyo, Japan), Sato Pharmaceutical (Tokyo, Japan), Galderma (Tokyo, Japan), and Japan Space Forum. This study was financially supported by Galderma. The authors alone are responsible for the content and writing of the paper and declare no conflicts of interest The Societies and Wiley Publishing Asia Pty Ltd

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