In Vitro Susceptibility of Dermatomycoses

Microbiol Immunol 2014; 58: 1–8 doi: 10.1111/1348-0421.12109

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 Tamura1,2, Miwa Asahara1, Mikachi Yamamoto3, Mariko Yamaura3, Mitsuru Matsumura1, Kazuo Goto1, Ali Rezaei‐Matehkolaei4, Hossein Mirhendi5, Miho Makimura6 and Koichi Makimura1,3,6,7,8

1Department of Clinical Laboratory Science, Graduate School of Medical Technology, 2Eiken Chemical, 4‐19‐9 Taito, Taito‐ku, Tokyo 110‐ 8408, Japan, 3Laboratory of Space and Environmental Medicine, Graduate School of Medicine, 4Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Golestan Avenue, Ahvaz, 5Department of Medical Parasitology and Mycology, School of Public Health and National Institute of Health Research, Tehran University of Medical Sciences, Tehran 14155‐6446, Iran, 6Teikyo University Institute of Medical Mycology, 7Department of Biomedicine, General Medical Education Center and 8Asia International Institute of Infectious Disease Control, Teikyo University, 2‐11‐1, Kaga, Itabashi, Tokyo 173‐8605

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, Trichophy- ton rubrum.

Correspondence Koich Makimura, Laboratory of Space and Environmental Medicine, Graduate School of Medicine, Teikyo University, 2‐11‐1 Kaga, Itabashi‐ku, Tokyo 173‐8605, Japan. Tel: þ81 3 3964 2140; fax: þ81 3 3964 8415; email: [email protected] Received 2 August 2013; revised 25 October 2013; accepted 28 October 2013.

List of Abbreviations: CLSI, Clinical Laboratory Standard Institute; FIC, fractional inhibitory concentration; MIC, minimum inhibition concentration.

A group of fungi that infect keratinized tissues (skin, hair, (TIMM2789, T. mentagrophytes (Arthroderma vanbreuseghe- and nails) of humans and animals cause dermatomy- mii)) and 43 clinical isolates of major pathogenic coses, including tinea. The major dermatophytes that dermatophytes were used; namely, 14 strains of T. rubrum, cause tinea are Trichophyton rubrum, Trichophyton menta- 14 strains of T. mentagrophytes human type (18) (synonym, grophytes, Trichophyton verrucosum, Microsporum canis, Micro- Trichophyton interdigitale (anthropophilic)) (19), three strains sporum gypseum and Epidermophyton floccosum. In addition, of Trichophyton tonsurans, one strain of T. verrucosum,two Candida spp. and non‐dermatophytic molds have also strains of M. canis,fourstrainsofM. gypseum and five strains been reported as causes of dermatomycosis (1). of E. floccosum. In addition, 10 strains of non‐dermatophytes Several antifungal agents have been developed and used were also used; namely, two strains of Aspergillus fumigatus, for internal and/or external treatment of dermatomycoses. two strains of Geotrichum candidum,twostrainsof Azole antifungal agents, such as ketoconazole, itracona- Scopulariopsis brevicaulis, two strains of Fusarium oxysporum, zole and bifonazole, inhibit lanosterol 14a‐demethylase one strain of Fusarium verticillioides and one strain of and block fungal membrane ergosterol biosynthesis in the Fusarium solani.Allisolateswereidentified using a cell (1, 2). The non‐azole antifungal agent, amorolfine, molecular‐basedmethodreportedpreviously(18–21). blocks other pathways of D14‐sterol reductase and D7– D8‐steroid isomerase in fungal cells (3). Terbinafine, an Preparation of isolates allylamine, inhibits fungal squalene epoxidase (1). Itra- The test isolates were subcultured onto 1/10 Sabouraud conazole and terbinafine have been approved in the USA dextrose agar (peptone, 1 g; glucose, 4 g; distilled water, and amorolfine and fluconazole have been approved in 1 L; agar, 15 g; pH 6.0) plates and incubated at 30°C for Europe for treatment of onychomycoses (2). 7 days. Some poor growth strains were cultivated for Onychomycoses are often recurrent, chronic, and extended times of up to 14 days. generally require long‐term treatment with antifungal agents (4). It is desirable to choose appropriate antifungal Antifungal agents drugs in the early stages of infection. In addition, it is practical to consider appropriate combinations of internal The following six antifungal agents were assessed: and external antifungal drugs with different pharmaco- amorolfine, terbinafine, butenafine, ketoconazole, itra- logical effects to treat refractory fungal infection, conazole, and bifonazole (Wako Pure Chemical Indus- especially onychomycosis. There have been many previous tries, Osaka, Japan). The antifungal drugs were dissolved studies of double or triple drug combination therapy (3– in dimethylsulfoxide. According to CLSI protocol M38‐ 17). These reports suggest the usefulness of combinations A2 (22), serial twofold dilutions were prepared with of external and internal antifungal agents; however, there powdered RPMI‐1640 medium (Gibco, Grand Island, have been few reports presenting quantitative data NY, USA) and buffered with 3‐(N‐morpholino)propane- regarding drug combinations in vitro (6, 7, 9). sulfonic acid at pH 7.0. to reach final concentrations of Here, we investigated the susceptibility of major 0.03–16 mg/mL for amorolfine, 0.001–0.5 mg/mL for dermatophytes and non‐dermatophytic fungi responsible terbinafine, 0.001–0.5 mg/mL for butenafine, 0.015– for superficial fungal infection to six antifungal agents: 8 mg/mL for ketoconazole, 0.015–8 mg/mL for itracona- amorolfine, terbinafine, butenafine, ketoconazole, itra- zole and 0.12–64 mg/mL for bifonazole with RPMI 1640 conazole and bifonazole. test medium. To calculate the FIC index, a checkerboard We also investigated the synergistic or additive effect of was designed with amorolfine (0.015–8 mg/mL) and an antifungal combination. We choose two antifungals in itraconazole (0.015–1 mg/mL). common use, amorolfine and itraconazole, which have different mechanisms of actions and administration routes Inoculum preparation (amorolfine is an external agent for topical use and The subcultured isolates were collected with sterile swabs itraconazole an internal agent for systemic use). We used and suspended in 2 mL of sterile 0.85% saline. Conidia the FIC index to quantify the efficacy of a combination of suspensions were filtered with sterile gauze and the amorolfine and itraconazole in 27 strains of dermatophytes. concentrations quantified with a hemocytometer to adjust to McFarland No. 1 (106 conidia/mL). MATERIALS AND METHODS Minimum inhibitory concentration tests Strains Antifungal susceptibility tests were performed by a The strains investigated in this study are shown in Table 1 broth microdilution method according to modified CLSI (Cl‐I‐ and Sz‐k‐ were clinical isolates). One standard strain protocol M38‐A2 (22). Briefly, aliquots of 100 mLofeach

Table 1. Strains assessed

Dermatophytes Non‐dermatophytes

Species Strains Species Strains

Trichophyton rubrum Cl‐I‐488 Aspergillus fumigatus ATCC 26430 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 31631 Cl‐I‐729 Fusarium solani TSY 04303 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 control well, this assessment being made on a visual microplates and stored at 70°C until use. Conidia basis (22–29). suspension was diluted tenfold with sterile saline and 2 mLinoculatedinto100mL of RPMI1640 test medium. Cumulative percentage curves of The microplates were incubated at 30°C for 3–7daysuntil dermatophytes the drug‐free control well was fully occupied by fungal growth. The MIC was deﬁned as the minimal concentra- Cumulative MIC percentage curves were used to permit tion required to inhibit 80% of the growth in the drug‐free visual analysis of MIC distribution (30). Cumulative

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) 0.12–0.5 0.004–0.06 0.015–0.12 0.06–0.5 0.015–0.25 0.12–1 T. mentagrophytes (n ¼ 16) 0.12–0.5 0.03–0.06 0.06–0.12 0.5–2 0.015–0.5 0.5–4 T. tonsurans (n ¼ 3) 0.25 0.015–0.06 0.12 0.5–2 0.06–0.25 0.5–2 T. verrucosum (n ¼ 1) 0.12 0.015 0.12 0.12 0.12 0.25 M. canis (n ¼ 2) 0.06–0.25 0.008–0.03 0.12 0.25–0.5 0.015–0.03 1–2 M. gypseum (n ¼ 4) 0.12–0.25 0.004–0.06 0.06–0.12 0.5–4 0.06–1 0.12–8 E. floccosum (n ¼ 5) 0.25 0.015–0.03 0.06 0.25–0.5 0.03–0.5 0.25–0.5 percentage curves of six antifungal agents for T. rubrum, agents for dermatophytes are listed in Table 2 and those T. mentagrophytes and 44 strains of clinically isolated for non‐dermatophytes in Table 3. The six antifungal dermatophytes were calculated. agents inhibited growth of dermatophytes, but showed markedly higher and wider MIC distribution in non‐ Calculation and evaluation of FIC index dermatophytes. In particular, Fusarium spp. were insensi- tive to all anti‐dermatophytic agents assessed in this Reading and interpretation of the results of combination study. examinations were performed in accordance with the The cumulative MIC percentage curves of the six method of Santos et al. (9). The interactions between antifungal agents for dermatophytes are shown in antifungal agents (drugs A and B) were quantitatively Figure 1. For two major causes of dermatomycoses, T. evaluated by the FIC index, which was calculated rubrum and T. mentagrophytes, MIC ranges of non‐azole according to the formula (MIC of A in combination/ agents were narrower than those of azole agents. The MIC of A) þ (MIC of B in combination/MIC of B). The MICs of total dermatophytes showed the same tendency interaction was deﬁned as synergistic if the FIC index was (solid line). Unexpectedly, there were marked differences 0.5, additive if it was >0.5 but 1, no interaction if it between T. rubrum and T. mentagrophytes in the MIC ranges was 2 and antagonistic if it was >2. of ketoconazole and bifonazole.

RESULTS Results for the combination of amorolfine Minimum inhibitory concentrations of and itraconazole antifungal agents in isolates Table 4 presents a summary of the FIC indexes of 27 All isolates grew in 1/10 Sabouraud agar after 3–14 days of clinical dermatophyte isolates. Synergistic interactions incubation. Aspergillus spp. and Fusarium spp. Grew were observed in 7 of 27 strains with FIC indexes of 0.5, relatively quickly (about 3 days) and T. rubrum and additive interactions in 16 isolates with FIC indexes >0.5 Microsorum spp. relatively slowly (7–14 days). After 1 and four isolates had FIC indexes of 2 (no genomic identification, each isolate was subjected to interaction). In total, the combination of amorolfine MIC assay. The MIC values of the six assessed antifungal 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 0963 2 0.12 >0.5 4 1 >64 TIMM 0697 1 0.25 >0.5 2 1 >64 Scopulariopsis brevicaulis TSY 0668 0.25 0.12 0.5 4 >82 NBRC 4843 0.25 0.12 >0.5 2 >84 Aspergillus fumigatus ATCC 26430 >64 >0.5 >0.5 8 1 4 TIMM 3968 >64 >0.5 >0.5 8 0.5 2 Fusarium oxysporum TSY 0351 >64 >0.5 >0.5 >8 >8 >64 NBCB 31631 >64 >0.5 >0.5 >8 >8 >64 Fusarium solani TSY 04303 8 >0.5 >0.5 >8 >8 >64 Fusarium verticillioides TSY 0219 >64 >0.5 >0.5 2 2 >64

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

Table 4. FIC indexes of dermatophytes We observed a synergistic effect in 7 of 27 strains with FIC indexes 0.5. Using a checkerboard method, Santos et al. Species Strains FIC index Evaluation demonstrated synergistic interactions between azoles and T. rubrum Cl‐I‐705 0.49 Synergistic cyclopiroxamine against T. rubrum and T. mentagrophytes (9). Cl‐I‐760 0.49 Harman et al.alsoreportedasynergisticeffect(1) of a ‐ ‐ Cl I 651 0.6 Additive combination of amorolfine and itraconazole in 46% of all Cl‐I‐488 0.605 organisms tested, including dermatophytes and non‐ Cl‐I‐726 0.625 Cl‐I‐729 0.625 dermatophytes(6).Inthepresentstudy,weusedastricter Cl‐I‐714 0.72 criterion for determination of synergy (0.5) and Cl‐I‐733 0.72 confirmed that a combination of these drugs had a Cl‐I‐732 1 synergistic (0.5) effect in 25.9% of samples and an Cl‐I‐724 2 No interaction additive (FIC index 1and0.5) effect in 59.3% of ‐ ‐ Cl I 725 2 samples. In total, these agents showed additive or synergistic T. mentagrophytes Cl‐I‐710 0.24 Synergistic effects on more than 85% of the strains examined. In Cl‐I‐558 0.48 Cl‐I‐757 0.48 particular, we found additive or synergistic effects in 19 of Cl‐I‐764 0.48 21 Trichophyton strains (90%) and in three strains of M. Cl‐I‐723 0.62 Additive gypseum (100%). We identified no additive or synergistic Cl‐I‐743 0.62 effectsintwoofthreestrainsofE. floccosum and detected no Cl‐I‐744 0.62 antagonistic effects in any of the 27 dermatophytes. ‐ ‐ Cl I 703 1 These results suggest that the combination of these two T. tonsurans Cl‐I‐718 0.49 Synergistic drugs can be expected to act additively or synergistically T. verrucosum Cl‐I‐749 0.75 Additive M. gypseum Sz‐k‐1 0.74 Additive in the treatment of dermatomycoses. Further investiga- Cl‐I‐727 0.75 tion is required to examine the effects of antifungal drug Sz‐k‐2 0.98 combination against these and other clinically important E. floccosum Cl‐I‐701 0.74 Additive dermatophytes. Cl‐I‐740 2 No interaction Although several studies have examined the synergic ‐ ‐ Cl I 745 2 effects of antifungal agents (34, 35), few have provided FIC index 0.5 is synergistic, >0.5 to 1 additive, 2 no interaction and explanations for the mechanisms of drug synergy (36). >2 antagonistic. 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 Minimum inhibitory concentrations varied widely mechanisms of drug synergy between amorolfine and among non‐dermatophyte strains (Table 3). In particular, itraconazole, we need to profile changes in cellular all antifungal agents showed high MICs in Fusarium spp. environment after drug administration. The variation of susceptibility seen in dermatophytic and ‐ non dermatophytic fungi indicates the necessity to ACKNOWLEDGMENTS identify the causative fungi to enable appropriate selection of effective antifungal drugs in each case and The authors thank the participating laboratories and to avoid development of resistance (31–33). hospitals for their cooperation and for providing the Several strategies using single or plural antifungals have fungal isolates described in this report. been reported for treating refractory dermatomyco- – fi ses, (3 17). Amorol ne is effective in several dermato- DISCLOSURE phytoses, especially tinea unguium (1, 3, 5, 6); however, it is only used topically. For systemic use, itraconazole or K.M. has received research grants from the following terbinafine is generally available. Lecha et al. (3) and companies: Hisamitsu Pharmaceutical (Tokyo, Japan), Baran et al. (5) described satisfactory results using Seikagaku Biobusiness (Tokyo, Japan), Kaken Pharma- combinations of amorolfine and terbinafine or itraco- ceutical (Tokyo, Japan), Dai‐Nippon Sumitomo Phar- nazole, respectively, in vivo. maceutical (Tokyo, Japan), Sato Pharmaceutical (Tokyo, We selected amorolfine and itraconazole to investigate Japan), Galderma (Tokyo, Japan), and Japan Space combinations of antifungal drugs. The former is a non‐ Forum. This study was financially supported by azole agent that is used topically (externally) and the latter Galderma. The authors alone are responsible for the an azole drug that is used systemically (internally). Both content and writing of the paper and declare no conflicts agents are commonly used for dermatomycoses. of interest.

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