Antifungal Combinations in Dermatophytes

Dermatophytes are the most common cause of fungal infections worldwide, affecting millions of people annually. The emergence of resistance among dermatophytes along with the availability of antifungal susceptibility procedures suitable for testing antifungal agents against this group of fungi make the combinatorial approach particularly interesting to be investigated. Therefore, we reviewed the scientific literature concerning the antifungal combinations against dermatophytes. A literature search on the subject performed in PubMed yielded 68 publications: 37 articles referring to in vitro studies and 31 articles referring to case reports or clinical studies. In vitro studies involved over 400 clinical isolates of dermatophytes (69% Trichophyton spp., 29% Microsporum spp., and 2% Epidermophyton floccosum). Combinations included two antifungal agents or an antifungal agent plus another chemical compound including plant extracts or essential oils, calcineurin inhibitors, peptides, disinfectant agents, and others. In general, drug combinations yielded variable results spanning from synergism to indifference. Antagonism was rarely seen. In over 700 patients with documented dermatophyte infections, an antifungal combination approach could be evaluated. The most frequent combination included a systemic antifungal agent administered orally (i.e., terbinafine, griseofulvin, or azole—mainly itraconazole) plus a topical medication (i.e., azole, terbinafine, ciclopirox, amorolfine) for several weeks. Clinical results indicate that association of antifungal agents is effective, and it might be useful to accelerate the clinical and microbiological healing of a superficial infection. Antifungal combinations in dermatophytes have gained considerable scientific interest over the years and, in consideration of the interesting results available so far, it is desirable to continue the research in this field.


Introduction
Dermatophytes are the most common cause of fungal infections worldwide, affecting millions of people annually. Dermatophytes are filamentous fungi with the ability to invade keratinised tissue, such as skin, hair, and nails [1]. Classically, they are divided into three genera: Trichophyton, Epidermophyton, and Microsporum [2]. However, this classification is based on the phenotype of the species and led to misclassification of morphological mutants. In 2017, de Hoog et al. constructed a phylogenetic tree using sequences of the nuclear ribosomal internal transcribed spacers (ITS rDNA) and divided the dermatophytes into seven clades: Trichophyton, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, Microsporum, and Arthroderma [3]. Based on their host specificity, these fungi are classified into three ecological groups: geophilic, zoophilic, and anthropophilic. Geophilic dermatophytes rarely cause infection in animals and humans but may be carried by animals in their fur. Zoophilic dermatophytes occur in the fur of animal hosts, either symptomatically or asymptomatically, and can be easily transmitted to humans. When zoophilic and geophilic species are transmitted to humans, they cause acute, inflammatory mycoses. Transmission of anthropophilic dermatophytes is usually from human to human. They cause chronic, mild, noninflammatory infections [4,5]. Ringworm or tinea is one of the most frequent clinical aspect of dermatophytosis. Among the tinea infections, tinea corporis, tinea cruris, tinea pedis, and onychomycosis are the most predominant types. The dermatophytes T. rubrum, T. interdigitale and T. mentagrophytes, are the main aetiological agents of dermatophytosis of skin and nails in humans [1][2][3][4][5].
Medical treatment of dermatophytosis consists of topical or oral antifungal agents. There are many topical agents for treating several less severe forms of tinea [6]. The azole derivatives, such as clotrimazole, miconazole, econazole, and oxiconazole, are the generally used. Agents from the allylamine family, such as terbinafine and naftifine, are also used. Other topical agents, such as ciclopirox or amorolfine, can be effective in the less severe cases of onychomycosis. In the more severe forms of dermatophyte infections, oral treatment is generally employed [6]. The first oral agent used to treat a dermatophyte infection was griseofulvin, introduced in clinical practice in 1958 [7]. This molecule interferes with microtubule formation, thus impairing fungal growth and cell division. Allylamines (mainly terbinafine) and triazoles (mainly itraconazole) are used for oral therapy. Both allylamines and triazoles act on the same cellular target, that is, the cell membrane. Triazoles inhibit sterol 14-α-demethylase, and allylamines inhibit squalene epoxidase, the inhibition of both enzymes leading to inhibition of ergosterol biosynthesis. Allylamines also lead to the accumulation of lanosterol, a toxic intermediary compound of the ergosterol biosynthesis pathway [8][9][10]. Terbinafine, which acts as a fungicide, is the drug of choice against Trichophyton spp. because of its clinical efficacy [11]. However, in the last years, an increasing incidence of chronic and recalcitrant dermatophytic infections have been described. Although rare, resistance to terbinafine has been documented among isolates of T. rubrum and T. mentagrophytes/T. interdigitale complex [12]. The resistance is generally due to several point mutations in the squalene target gene. This phenomenon, first described in recalcitrant dermatophytosis observed in India, was later reported in other countries [12][13][14][15][16][17]. Due to a very limited number of antifungals effective against dermatophytes and the emergence of resistance to these drugs, an in vitro antifungal susceptibility testing should be implemented in reference laboratories to monitor this phenomenon.
Currently, two standardized techniques for in vitro antifungal susceptibility testing of dermatophytes based on a broth microdilution procedure are available: one from the Clinical Laboratory Standards Institute (CLSI) and the other from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [18,19]. Although very similar, the two methods differ in endpoint determination. Lately, the EUCAST method was validated in a multicentre study (10 laboratories) in which terbinafine, itraconazole, voriconazole, and amorolfine were tested against a blinded panel of 38 terbinafine wild types and target gene mutant isolates of T. rubrum and T. interdigitale. The higher interlaboratory reproducibility was obtained using a medium with the addition of chloramphenicol and cycloheximide and measuring the MIC spectrophotometrically at 50% inhibition [20].
An antifungal combination strategy has been lately implemented to overcome the resistance phenomenon against a wide variety of infections due to either yeasts or filamentous fungi [21,22]. Achievement of a synergistic interaction is desirable in these contexts. The emergence of resistance among dermatophytes along with the availability of antifungal susceptibility procedures suitable for testing antifungal agents against this group of fungi make the combinatorial approach particularly interesting to be investigated. Therefore, we aimed to review the scientific literature concerning the antifungal combinations used against dermatophytes. In order to include most of the published papers on this topic, the revision was carried out using the classic dermatophyte nomenclature, which divides these fungi into three genera. In particular, the results of in vitro combinations of several antifungals or antifungals plus other chemical compounds are presented. Additionally, the effects of combinatorial regimens in human infections are reported.

Materials and Methods
This systematic review was conducted in accordance with the PRISMA guidelines [23] ( Figure 1). PubMed was searched for dermatophytes antifungal combinations therapy with the following search string: "trichophyton" and "antifungal" and "combination"; "microsporum" and "antifungal" and "combination"; "epidermophyton" and "antifungal" and "combination". Literature search was conducted on 1 June 2021, by three individual researchers (L.B., S.F., and G.M.). In case of discrepancies in the process of inclusion of papers/data extraction, a consensus was reached through discussion or involvement of a fourth reviewer (F.B.). Additional cases were sought from the reference list of included papers. The inclusion criteria were antifungal combinations for Trichophyton spp., Microsporum spp., and Epidermophyton floccosum. The exclusion criteria were papers not referring to human studies (i.e., veterinary cases), papers in languages other than English, unreachable publications, papers not specifying the genera/species of dermatophytes, reviews of the literature, combinations considering two chemical compounds other than antifungals, and combinations not considering chemical compounds (i.e., photodynamic therapy). Data from the included papers were entered in a database, created with Excel, which encompassed the genus/species/number of dermatophytes tested, the type of drug combination, the method utilized for testing, and the results of the interaction. In case of clinical reports, demographic data (when available) and outcome of the combination therapy were also reported.

Materials and Methods
This systematic review was conducted in accordance with the PRISMA guidelines [23] (Figure 1). PubMed was searched for dermatophytes antifungal combinations therapy with the following search string: "trichophyton" and "antifungal" and "combination"; "microsporum" and "antifungal" and "combination"; "epidermophyton" and "antifungal" and "combination". Literature search was conducted on 1 June 2021, by three individual researchers (L.B., S.F., and G.M.). In case of discrepancies in the process of inclusion of papers/data extraction, a consensus was reached through discussion or involvement of a fourth reviewer (F.B.). Additional cases were sought from the reference list of included papers. The inclusion criteria were antifungal combinations for Trichophyton spp., Microsporum spp., and Epidermophyton floccosum. The exclusion criteria were papers not referring to human studies (i.e., veterinary cases), papers in languages other than English, unreachable publications, papers not specifying the genera/species of dermatophytes, reviews of the literature, combinations considering two chemical compounds other than antifungals, and combinations not considering chemical compounds (i.e., photodynamic therapy). Data from the included papers were entered in a database, created with Excel, which encompassed the genus/species/number of dermatophytes tested, the type of drug combination, the method utilized for testing, and the results of the interaction. In case of clinical reports, demographic data (when available) and outcome of the combination therapy were also reported.

Results and Discussion
A total of 205 articles were initially identified ( Figure 1). After duplication removal, 166 articles were screened. Further exclusion included papers that were out of topic (34), veterinary (8), not in English (19), without fungal identification (3), literature reviews (14), and about combinations not including at least one antifungal agent (20). Additional 7 papers found in the reference list of the screened articles were added to the 61 eligible papers. Therefore, a total of 68 publications were included in this review: 37 articles referring to in vitro studies, and 31 articles referring to case reports or clinical studies (Tables 1-3). Among the first group of articles, there were 7 reports in which the combination of two antifungal agents was used, while 30 articles in which an antifungal agent was combined with a chemical compound other than an antifungal agent.

Antifungal Combinations
The results of in vitro antifungal combinations are reported in Table 1. Trichophyton spp. represented the most common genus tested, followed by Microsporum spp. and E. floccosum. Combinations included amorolfine plus azoles or terbinafine or griseofulvin; azoles plus griseofulvin or terbinafine; azoles plus ciclopirox [24][25][26][27][28][29][30]. Checkerboard titration methodology was the most common procedure for testing a combination (6/7 studies). Two studies investigating the effects of the combination of amorolfine or ciclopirox plus azoles found 100% synergistic interaction against many Trichophyton spp. [26,28]. One study confirmed this positive effect by adding two additional methods (disk-diffusion and E-test assays) to the broth microdilution procedure [26]. Although antagonism was never observed in any report, the type of interaction varies according to drug and isolate tested. In general, amorolfine plus azoles yielded synergistic interaction more often than amorolfine plus griseofulvin or plus terbinafine. One study investigated three new topical drugs (efinaconazole, tavaborole, and luliconazole) with itraconazole or terbinafine against T. rubrum and T. interdigitale. Efinaconazole with terbinafine or itraconazole exerted a synergistic effect on 43.8% and 12.5% of the strains tested, respectively. Conversely, luliconazole showed no synergistic effect with terbinafine but was synergistically effective with itraconazole against 31.3% of the strains. Tavaborole (an inhibitor of protein synthesis in fungal cells) showed no synergistic effect with terbinafine and was synergistically effective with itraconazole against 18.8% of the strains [29].
Overall, these data would suggest that an antifungal combination regimen might be useful against an infection due to dermatophytes. It is interesting to note that even combining drugs acting against a common fungal target (i.e., ergosterol-azoles, allylamines, and morpholine drugs such as amorolfine), a positive interaction in terms of reduction of the MIC of both drugs is often observed.

Antifungals Combined with Several Chemical Compounds
The results of in vitro activities of antifungals combined with other compounds are reported in Table 2. Again, Trichophyton spp. represented the most common genus tested. Combination included: azoles or terbinafine or griseofulvin plus plant extracts including essential oils (19/30 studies), azoles or terbinafine or amorolfine plus immunosuppressant agents (3 studies), azoles or terbinafine plus peptides (3 studies), azoles plus disinfectants (3 studies), and other combinations including antifungal agents plus efflux pump inhibitors and statins. Checkerboard titration methodology was the most common procedure for testing the combination, followed by agar methods (i.e., disk diffusion and agar dilution) .
It has been shown that plants have the capacity to produce secondary metabolites, including those which are constituents of essential oils, as defence mechanisms against herbivores and microorganisms. They act in two ways: by neutralizing free radicals (the antioxidant effect) and as anti-inflammatory agents by inhibiting the release of proinflammatory mediators. Secondary metabolites produced by plants are also capable of acting in a third way, as antifungal agents [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]. A synergistic interaction between antifungal agents and natural products was often seen ( Table 2). One recent study evaluated the antifungal activity of tea tree oil (TTO) (Melaleuca alternifolia essential oil) and its main components against T. rubrum alone and in association with ketoconazole or itraconazole and showed either their fungicidal effects or a synergism upon combination with azoles [49]. Most of the studies demonstrated that the type of interaction was either isolate-or drugdependent. One research assessed the antifungal activity of essential oil from Mentha x piperita against a wide panel of dermatophyte clinical isolates and found a fungistatic activity against these fungi. When this compound was used in combination with azoles, a synergic interaction was observed for T. mentagrophytes while indifference was detected for T. rubrum and M. canis [48]. Overall, these data would suggest that these natural compounds are one of the most promising sources for pharmacological research and that the development of new natural antimicrobial agents against many microbial pathogens, including dermatophytes, is warranted.
Calcineurin inhibitors (i.e., tacrolimus and cyclosporin A) or inhibitors of the mTOR pathway (i.e., sirolimus) are anti-rejection drugs widely used in organ transplant recipients and to prevent graft-versus-host disease in allogeneic stem cell recipients. However, these compounds also possess intrinsic antifungal activity against selected fungi [50][51][52]. One study evaluated the in vitro interactions between tacrolimus or triamcinolone acetate with itraconazole, terbinafine, bifonazole, and amorolfine against 28 clinical dermatophyte isolates, including 13 T. rubrum, 6 T. mentagrophytes, 5 M. canis, and 4 E. floccosum and found that a synergistic interaction was more often observed when the antifungal agents were combined with tacrolimus rather than cortisone [52]. Another study evaluated the combination of fluconazole with either tacrolimus or cyclosporine in an ex vivo T. mentagrophytes human skin infection model. Conidia colonization was monitored by scanning electron microscopy over a 7-day treatment period. The fluconazole-tacrolimus combination was superior to one single-drug therapy by clearing conidia and protecting skin from damage at low drug concentrations [50]. Similarly, when tacrolimus was added to itraconazole against 5 isolates of T. mentagrophytes, a synergistic interaction was observed in 80% of the cases [51]. Overall, these data indicate that calcineurin inhibitors are synergistic with ergosterol biosynthesis inhibitors against dermatophytes, and that a potential clinical application may be desirable.   Another interesting therapeutic approach might be represented by peptides because they act efficiently and rapidly against a wide range of pathogens including bacteria, fungi, viruses, and protozoa. Moreover, peptide resistant mutants rarely emerge with these molecules, especially when they are used in combination with other anti-infective drugs [53][54][55]. Among these compounds, protegrins and defensin were originally iso-lated from mammalian leucocytes. One study evaluated the in vitro effects of IB-367 alone and in combination with three antifungal drugs against 20 clinical isolates of dermatophytes belonging to three species and showed synergism in 35%, 30%, and 25% of IB-367/fluconazole, IB-367/itraconazole, and IB-367/terbinafine interactions, respectively. IB-367 exerted a fungicidal activity against T. mentagrophytes, T. rubrum, and M. canis at concentrations starting from 1 × MIC. At a concentration of 5 × MIC, IB-367 showed the highest rates of hyphae damage for M. canis and T. mentagrophytes [55]. Another study investigated the in vitro effects of tachyplesin III (TP), a potent disulphide-linked peptide, in combination with terbinafine against 20 clinical isolates of dermatophytes belonging to four species. Terbinafine in combination with TP showed indifferent activity against 14 of the 20 isolates (70%); synergic activity against 6 of them (30%); no antagonistic activity was observed [54]. Finally, the lipopeptide Pal-Lys-Lys-NH 2 (PAL) alone and in combination with standard antifungal agents was tested against 24 clinical isolates of dermatophytes belonging to four species. Synergy was observed in 67%, 52%, and 15% of PAL/itraconazole, PAL/terbinafine, and PAL/fluconazole interactions, respectively. None of these combinations yielded antagonistic interactions. When synergy was not achieved, there was still a decrease in the MIC of one or both drugs used in the combination [53]. Overall, these studies demonstrated that peptides have potential activity against dermatophytes. These drugs, applied in the form of lacquer, spray, or ointment, could represent an interesting new therapy, particularly when combined with conventional treatment in recalcitrant or resistant dermatophyte infections.
Another combinatorial approach investigated the activity of an antifungal, generally miconazole, with the antiseptic compound chlorhexidine [56][57][58]. One study demonstrated that this association yielded a synergistic effect in vitro against 5 out of 10 isolates of M. canis, and an additive effect against 4 isolates, while when the same combination was tested against 9 isolates, each of T. mentagrophytes and T. erinacei, the most frequent interactions observed were additivism or indifference. Again, antagonism was never observed [57,58].
In general, the results obtained by combination of antifungal agents with chemical compounds other than antifungals yielded variable results spanning from synergism to indifference. Antagonism was rarely seen. This interaction is well documented for natural products (i.e., essential oils) as shown by a substantial number of scientific publications. Although promising results were documented, the different methods used to test these combinations hampered a univocal and comprehensive conclusion on the real effectiveness of these combinations.

Clinical Cases
The results of antifungal combinations in humans are reported in Table 3. There were 25 papers describing 37 single case reports, one paper each describing 36 and 254 patients, respectively, and 3 clinical trials involving a total of 410 patients . Either paediatric or adult patients were represented. Tinea corporis, tinea capitis, and tinea unguium were the most common clinical manifestations. In the single case reports, the most frequent combination approach included a systemic antifungal agent administered orally (i.e., terbinafine, griseofulvin, or azole-mainly itraconazole) plus a topical medication (i.e., azole, terbinafine, ciclopirox, cortisone) for several weeks. Few cases were treated with both drugs given topically or orally. Only in two clinical cases, resistance mechanisms were assessed and confirmed by sequencing of the SQLE gene [67,70]. One patient with Trichophyton endophthalmitis and five patients with fungal keratitis due to T. shoenleinii were treated with a combination of systemic antifungal agents, including voriconazole or fluconazole, plus an antifungal agent administered topically (amphotericin B or miconazole). The outcome consisted in full recovery or improvement in most of the cases [74,86]. The 36 patients included in one paper consisted of 18 children and 18 adults with infections due to T. violaceum. The source of contagion was traced to 13 children, 11 African and 2 Ukrainian, adopted from an orphanage, with misdiagnosed tinea capitis. All 13 index cases and the 16 patients infected by them were treated with griseofulvin for 45 days and topical imidazoles. The adults with spreading tinea corporis were treated with 100 mg itraconazole for 15-20 days and those with tinea capitis with the same dose of the antimycotic for 45 days and with topical imidazoles. In all patients, recovery was confirmed by clinical and mycological examination 3 months after healing [87]. One early observational study involving 254 patients with various forms of dermatophyte infections mainly due to Trichophyton spp., concluded that topical treatment (Wilkinson's salve, iodized alcohol 5%, undecylenic acid derivatives, 5-bromo-4 -chlorosalicylanilides, tolnaftates) plus griseofulvin possibly enhances the healing capacity and shortens the time for treatment, but it has no effect in preventing reinfections [88]. One randomized study of toenail onychomycosis with matrix area involvement due to T. rubrum in most cases, compared amorolfine 5% nail lacquer once weekly for 24 weeks given with 200 mg of itraconazole once daily for 6 or 12 weeks vs. itraconazole alone given for 12 weeks [91]. Combination therapy showed to be significantly more effective than monotherapy, both in terms of mycological and clinical cures at week 12. Similarly, another randomized study comparing amorolfine plus terbinafine vs. terbinafine alone in 249 patients with onychomycosis showed a significantly higher success rate for patients undergoing combination therapy relative to those in monotherapy at 18 months [89]. Another randomized study investigated the efficacy of combination therapy with oral griseofulvin and oral prednisolone to oral griseofulvin alone in the treatment of kerion celsi due to Trichophyton spp. [91]. Both groups were treated with oral griseofulvin for 8 weeks, whereas oral prednisolone was given in tapering doses for 3-4 weeks to the first group only. The final evaluation at week 12 showed a cure rate of 100% in both groups without any significant difference in terms of clinical or mycological cure.   Topical treatment plus GRI possibly enhances the healing capacity and shortens the time of treatment but no effect in the recurrences

Conclusions
Although dermatophyte infections are rarely life threatening, their chronicity and the frequency of relapse require prolonged treatment, resulting in an increased risk of drug toxicity and development of drug resistance. Similarly to what has been already observed in systemic fungal infections sustained by Candida spp. or Aspergillus spp., emergence of drug resistant strains among isolates of Trichophyton spp. has been lately documented.
Although dermatophytes are a group of fungi quite difficult to test in vitro (i.e., slow growth, inoculum preparation, incubation intervals etc.), standardized procedures have been introduced and validated, thereby making antifungal susceptibility testing of dermatophytes easier. This has led to experimenting with various pharmacological associations aimed at increasing the efficacy of the therapy against this group of fungi. Most of in vitro studies investigated the combination of classic antifungal agents with several, disparate, chemical compounds. The association between an antifungal drug and plant extracts, including essential oils, seems to evoke a particular interest. The reciprocal potentiation of the molecules upon combination makes these approaches particularly appealing in clinical practice. Although the intrinsic mechanisms of antifungal activity of these natural products have not been fully investigated, several cell targets are simultaneously involved, thereby making the occurrence of resistance unlikely. Clinical data indicate that an association of antifungal agents (systemic plus topic) is effective, and it might be useful in speeding up the clinical and microbiological healing of a superficial infection. It must be noted, however, that there are few controlled/randomized clinical trials and that unequivocal conclusions cannot be drawn. Another limitation is the lack of well-characterized antifungal-resistant isolates, whose treatment could especially benefit from a combination approach.
In summary, antifungal combinations against dermatophytes have gained considerable scientific interest over the years. To establish whether this approach can become a reliable treatment option, additional in vitro and clinical data are warranted.