Antifungal activity of ribavirin used alone or in combination with fluconazole against Candida albicans is mediated by reduced virulence

The incidence of fungal infections has increased continuously in recent years, and drug resistance, especially resistance to fluconazole (FLC), has emerged. To overcome this challenge, research on the antifungal activities of non-antifungal agents has gained more attention. In this study, we determined the anti-Candida activity of ribavirin (RBV), an antiviral drug commonly used in the clinic, and found that RBV displayed potent antifungal activity when used alone or in combination with FLC in vitro and in vivo. In vitro, the MIC₈₀ values of RBV were 2–4 µg/mL for FLC-susceptible Candida albicans and 8 µg/mL for FLC-resistant C. albicans. When RBV at a dose of 1 µg/mL was combined with FLC, significant synergistic effects were exhibited against FLC-resistant C. albicans, and the MICs of FLC decreased from >512 µg/mL to 0.25–1 µg/mL. Synergism was also exhibited against C. albicans biofilms. In vivo, RBV plus FLC significantly improved the survival of infected Galleria mellonella larvae compared with the FLC-treated group over a 4-day period and attenuated the damage of FLC-resistant C. albicans to G. mellonella larvae tissue. Furthermore, mechanistic studies indicated that the antifungal effects of RBV used alone or in combination with FLC might be associated with inhibition of biofilm formation, reduced extracellular phospholipase activity and inhibition of hyphal growth, but is not related to promotion of FLC uptake and inhibition of FLC efflux. These results provide a promising direction for overcoming drug resistance and for expanding the clinical application of existing drugs.     

Combination of fluconazole with non-antifungal agents: A promising approach to cope with resistant Candida albicans infections and insight into new antifungal agent discovery

The past decades have witnessed a dramatic increase in invasive fungal infections, especially candidiasis. Despite the development of more effective new antifungal agents, fluconazole (FLC) is still widely used in the clinic because of its efficacy and low toxicity. However, as the number of patients treated with FLC has increased, FLC-resistant Candida albicans isolates emerge more frequently. In addition, biofilm-associated infections are commonly encountered and their resistance poses a great challenge to antifungal treatment. Various approaches have been proposed to increase the susceptibility of C. albicans to FLC in order to cope with treatment failures, among which is the combination of FLC with different classes of non-antifungal agents such as antibacterials, calcineurin inhibitors, heat shock protein 90 inhibitors, calcium homeostasis regulators and traditional Chinese medicine drugs. Interestingly, many of these combinations showed synergistic effects against C. albicans, especially resistant strains. The main mechanisms of these synergistic effects appear to be increasing the permeability of the membrane, reducing the efflux of antifungal drugs, interfering with intracellular ion homeostasis, inhibiting the activity of proteins and enzymes required for fungal survival, and inhibiting biofilm formation. These modes of action and the antifungal mechanisms of various compounds referenced in this paper highlight the idea that the reversal of fungal resistance can be achieved through various mechanisms. Studies examining drug interactions will hopefully provide new approaches against antifungal drug resistance as well as insight into antifungal agent discovery.     

Antifungal screening and in silico mechanistic studies of an in‐house azole library

Systemic Candida infections pose a serious public health problem with high morbidity and mortality. C. albicans is the major pathogen identified in candidiasis; however, non‐albicans Candida spp. with antifungal resistance are now more prevalent. Azoles are first‐choice antifungal drugs for candidiasis; however, they are ineffective for certain infections caused by the resistant strains. Azoles block ergosterol synthesis by inhibiting fungal CYP51, which leads to disruption of fungal membrane permeability. In this study, we screened for antifungal activity of an in‐house azole library of 65 compounds to identify hit matter followed by a molecular modeling study for their CYP51 inhibition mechanism. Antifungal susceptibility tests against standard Candida spp. including C. albicans revealed derivatives 12 and 13 as highly active. Furthermore, they showed potent antibiofilm activity as well as neglectable cytotoxicity in a mouse fibroblast assay. According to molecular docking studies, 12 and 13 have the necessary binding characteristics for effective inhibition of CYP51. Finally, molecular dynamics simulations of the C. albicans CYP51 (CACYP51) homology model's catalytic site complexed with 13 were stable demonstrating excellent binding.     

Analysis of global antifungal surveillance results reveals predominance of Erg11 Y132F alteration among azole-resistant Candida parapsilosis and Candida tropicalis and country-specific isolate dissemination

This study evaluated the activity of echinocandins, azoles and amphotericin B against Candida spp. isolates and other yeasts and characterised azole resistance mechanisms in Candida parapsilosis and Candida tropicalis. Invasive Candida spp. isolates (n = 2936) collected in 60 hospitals worldwide during 2016–2017 underwent antifungal susceptibility testing by broth microdilution. Azole-resistant C. parapsilosis and C. tropicalis were submitted to qPCR for ERG11, CDR1 and MDR1, and the whole genome sequence was analysed. Results of non-susceptibility to echinocandins ranged from 0.0–2.3%, being highest in Candida glabrata. More than 99.0% of the Candida albicans isolates were susceptible to both fluconazole and voriconazole. Fluconazole resistance in C. glabrata was 6.5% overall, being highest in the USA (13.0%). Resistance to voriconazole in Candida krusei was only noted in the USA (5.0%). Azoles inhibited 89.1–91.6% of C. parapsilosis isolates, with most resistant isolates noted in Europe (15.1%), including 36 isolates from Italy (three hospitals), of which 34 harboured Erg11 Y132F mutations and overexpressed MDR1. Azole non-wild-type C. tropicalis (7/227) were found in five countries: 3 isolates from Thailand had the same Erg11 Y132F alteration. Fluconazole non-wild-type isolates were noted among 3/77 (3.9%) Candida dubliniensis, 4/17 (23.5%) Candida guilliermondii, 4/47 (8.5%) Candida lusitaniae and other less common yeast species. Echinocandin use has been recommended over fluconazole for invasive Candida infections. However, azoles are still active against the most common Candida spp. and resistance appears to be restricted to certain geographic regions and associated with Erg11 Y132 alterations in C. parapsilosis and C. tropicalis.     

Anidulafungin,a new antifungal drug

ObjectivesThe development of new antifungal drugs has become a challenge for pharmaceutical industry due to the increasing incidence of invasive fungal infections.MethodsExperience of existing antifungal drugs has shown that, like in bacteria against antibiotics, most fungal species, especially in Candida spp, resistance to previously active drugs is developing and constitutes a major problem in treating invasive fungal infections.ResultsAmong series of newer drugs, experimental data and literature search have unravelled a new class of antifungals, Echinocandins which inhibit successfully Candida spp resistant to other drugs such as fluconazole. One drug, anidulafungin has emerged recently in this class as one of the most active in invasive fungal infections.     

Repurposing pantoprazole and haloperidol as efflux pump inhibitors in azole resistant clinical Candida albicans and non-albicans isolates

Candida species have a major role in nosocomial infections leading to high morbidity and mortality. Increased resistance to various antifungals, especially azoles is a significant problem. One of the main mechanisms for azole resistance is the up-regulation of efflux pump genes including CDR1 and MDR1. In the current study, clinical Candida isolates were identified to the species level and the antifungal susceptibility (AFS) of different Candida species was determined by disk diffusion method. Furthermore, the main mechanisms of azole resistance were investigated. Finally, haloperidol and pantoprazole were tested for their potential synergistic effect against fluconazole-resistant isolates.One hundred and twenty-two Candida clinical isolates were used in this study. 70 isolates were Candida albicans (57.4%), the non-albicans Candida species include: C. krusei (20.5%), C. tropicalis (6.6%), C. parapsilosis (5.7%), C. dubliniensis (4.9%) and C. glabrata (4.9%). The AFS testing showed that resistance to fluconazole and voriconazole were 13.1% (n = 16) and 9.8% (n = 12), respectively. Among the 16 resistant isolates, eight isolates (50%) were strong biofilm producers, seven (43.8 %) formed intermediate biofilm and one had no biofilm. All resistant strains overexpressed efflux pumps. Using RT-PCR, the efflux genes CDR1, MDR1 and ABC2 were over-expressed in azole resistant isolates. Haloperidol-fluconazole and pantoprazole-fluconazole combinations reduced the MIC of fluconazole in resistant isolates. The current study showed an increase in azole resistance of Candida species. The majority of resistant isolates form biofilm, and overexpress efflux pumps. Pantoprazole and Haloperidol showed a noteworthy effect as efflux pump inhibitors which oppose the fluconazole resistance in different Candida species.     

The antimicrobial effects of selenium nanoparticle-enriched probiotics and their fermented broth against Candida albicans

Background

Lactic acid bacteria are considered important probiotics for prevention of some infections. The aim of this work was to investigate the effect of selenium dioxide on the antifungal activity of Lactobacillus plantarum and L. johnsonii against Candida albicans.

Methods

Lactobacillus plantarum and L. johnsonii cells, grown in the presence and absence of selenium dioxide, and their cell-free spent culture media were tested for antifungal activity against C. albicans ATCC 14053 by a hole-plate diffusion method and a time-kill assay.

Results

Both L. plantarum and L. johnsonii reduced selenium dioxide to cell-associated elemental selenium nanoparticles. The cell-free spent culture media, from both Lactobacillus species that had been grown with selenium dioxide for 48 h, showed enhanced antifungal activity against C. albicans. Enhanced antifungal activity of cell biomass against C. albicans was also observed in cultures grown with selenium dioxide.

Conclusions

Selenium dioxide-treated Lactobacillus spp. or their cell-free spent broth inhibited the growth of C. albicans and should be investigated for possible use in anti-Candida probiotic formulations in future.     

Novel Carboline Derivatives as Potent Antifungal Lead Compounds: Design,Synthesis, and Biological Evaluation

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The echinocandins: three useful choices or three too many?

Echinocandins act by inhibiting 1,3-β-d-glucan synthesis in the fungal cell wall. The three licensed agents in this class, namely anidulafungin, caspofungin and micafungin, have a favourable pharmacological profile. These agents are narrow spectrum with clinically relevant activity against Candida and Aspergillus spp. Several trials have established the non-inferiority of these agents over existing agents in the treatment of invasive fungal infections. Caspofungin is also licensed for empirical antifungal therapy of presumed fungal infections in patients with febrile neutropenia. This paper reviews the literature on echinocandins.     

Nosocomial bloodstream infections due to Candida spp. in the USA: species distribution,clinical features and antifungal susceptibilities

Candida spp. are among the most frequent nosocomial pathogens, contributing significantly to morbidity and mortality. Longitudinal data on the epidemiology of Candida bloodstream infections (BSIs) are still limited. Isolates and clinical data from 1218 episodes of Candida BSI were prospectively collected from patients in 52 hospitals in the USA between 1998 and 2006. Susceptibilities to amphotericin B, flucytosine, fluconazole, posaconazole, voriconazole, anidulafungin, caspofungin and micafungin were determined for 1077 Candida isolates by the CLSI reference broth microdilution method using the recently published species-specific clinical breakpoints. Candida albicans was the most prevalent species (50.7%), followed by Candida parapsilosis (17.4%), Candida glabrata (16.7%) and Candida tropicalis (10.2%). The prevalence of non-albicans Candida spp. increased over time. Patients had a mean age of 51 years and a mean length of hospital stay prior to BSI of 22 days. The main underlying conditions were gastrointestinal (20.1%) and pulmonary (13.0%) diseases. Intravenous catheters (19.1%) and the urinary tract (8.0%) were the most frequently determined likely sources, whilst in the majority of patients (61.1%) no source could be identified. Overall mortality was 38.1%. Of the isolates studied, 0.8% of C. albicans, 100.0% of C. glabrata, 2.9% of C. parapsilosis and 4.9% of C. tropicalis were non-susceptible to fluconazole, and 0.6% of C. albicans, 5.0% of Candida krusei, 7.6% of C. parapsilosis and 9.8% of C. tropicalis were non-susceptible to voriconazole. All echinocandins showed good activity against most Candida spp., including the majority of C. parapsilosis isolates, but only 38.1% of C. glabrata tested susceptible to caspofungin.     

Arginolipid: A membrane-active antifungal agent and its synergistic potential to combat drug resistance in clinical Candida isolates

Antifungal drug resistance exhibits a major clinical challenge for treating nosocomial fungal infections. To find a possible solution, we synthesized and studied the antifungal activities of three different arginolipids (N^α-acyl-arginine ethyl ester) against clinical drug-resistant isolates of Candida. The most active arginolipid, oleoyl arginine ethyl ester (OAEE) consisting of a long unsaturated hydrophobic chain, was tested for its mode of action, which revealed that it altered ergosterol biosynthesis and compromised the fungal cell membrane. Also, OAEE was found to exhibit synergistic interactions with fluconazole (FLU) or amphotericin B (AmB) against planktonic Candida cells, wherein it reduced the inhibitory concentrations of these drugs to their in vitro susceptible range. Studies conducted against the C. tropicalis biofilm revealed that the OAEE+AmB combination synergistically reduced the metabolic activity and hyphal density in biofilms, whereas OAEE+FLU was found to be additive against most cases. Finally, the evaluated selective toxicity of OAEE toward fungal cells over mammalian cells could establish it as an alternative treatment for combating drug-resistant Candida infections.     

Impaired ergosterol biosynthesis mediated fungicidal activity of oil based tin polymer

Candida represents an important group of opportunistic fungal pathogens which causes infections in immunocompromised patients. The increasing emergence of fungal resistance to currently available antimycotic agents, especially azoles is a major problem. A vegetable-based organotin polymer synthesised by the condensation of fatty amide diol (N, N ¹-bis 2 hydroxy ethyl castor oil fatty amide) and butyl tin chloride dihydroxide has been reported to posses antimicrobial activity. This study is an attempt to investigate the antifungal activity of this compound against several fluconazole-susceptible and resistant clinical Candida isolates and to understand its mode of action. The susceptibility tests for the polymer were carried out in terms of minimum inhibitory concentrations (MICs) and disc diffusion assays against fifty-nine Candida isolates by employing standard protocols. The ergosterol-mediated antifungal activity was analysed by the sterol quantitation method. The organotin polymer was found to be effective, to varying extent, against all the Candida isolates including the resistant ones. It showed a low MIC value, which was fungicidal in contrast to the fungistatic fluconazole and caused considerable impairment of ergosterol biosynthesis in all the strains tested. This compound may be targeting the ergosterol biosynthesis pathway and may be used as an alternative to fluconazole which develops resistance during therapy. The selectively fungicidal characteristics of the organotin polymer indicate that this compound might be a promising antifungal agent.     

Antifungal resistance in non- albicans Candida species

Non- Candida albicans species have emerged as important bloodstream pathogens. They tend to have decreased susceptibility to antifungal agents in vitro and cause infections associated with high morbidity and mortality. Fluconazole resistance can emerge in any Candida spp., but is most commonly seen with Candida krusei, for which resistance is universal, and with Candida glabrata. Amphotericin B resistance has also been increasingly reported, most notably in isolates of Candida lusitaniae and Candida guilliermondii. Efforts are underway to correlate in-vitro antifungal susceptibility of individual Candida isolates with response to therapy of patients with candidemia. Future advances in this field might allow physicians to identify Candida isolates resistant to specific antifungal agents and thereby tailor therapy of candidemia.     

Benzo[g]quinazolines as antifungal against candidiasis: Screening,molecular docking,and QSAR investigations

Candida albicans, an opportunistic pathogen, is the most common type of fungus and represents a substantial source of human invasive disease (nosocomial infection). This category of fungi are part of our microbiota, and given the appropriate environmental conditions, it has the potential to cause both superficial and systemic infections. There is a soaring resistance against the available anticandidal agents. The purpose of this research is to investigate the activity of certain previously synthesized benzo[g]quinazolines against C. albicans in vitro by using the cup-plate diffusion method. There was a marked difference in the effectiveness of the target compounds 1–6 against the sample of C. albicans that was tested. Benzo[g]quinazolines 1 (inhibition zone = 20 mm) and 2 (inhibition zone = 22 mm) had good effects in comparison to fluconazole (inhibition zone = 26 mm). A docking study was conducted between benzo[g]quinazolines 1–6 and Candida spp. CYP51 to establish the binding mode compared with fluconazole and VT-1161 (oteseconazole) as reference medicines, and it was determined that binding at the active site of Candida spp. CYP51 occurred in the same manner. Quantitative structure–activity relationship (QSAR) investigation was performed to further characterize the identified anticandidal agents and recognize the major regulatory components governing such activity. In future studies, the benzo[g]quinazoline scaffold could serve as a model for the design and development of novel derivatives with antifungal potential.     

In vitro characterization,ADME analysis,and histological and toxicological evaluation of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains

BackgroundCandida species are one of the most common causes of nosocomial bloodstream infections among the opportunistic fungi. Extensive use of antifungal agents, most of which were launched on the market more than 20 years ago, led to the selection of drug-resistant or even multidrug-resistant fungi. We recently described a novel class of antifungal macrocyclic compounds with an amidinourea moiety that is highly active against azole-resistant Candida strains.ObjectiveA compound from this family, BM1, was investigated in terms of in vitro activity against various Candida species, including C. auris isolates, interaction with the ABC transporter, CDR6, and in vivo distribution and safety.MethodsIn vitro assays (CYP inhibition, microsomal stability, permeability, spot assays) were used to collect chemical and biological data; animal models (rat) paired with LC-MS analysis were utilised to evaluate in vivo toxicology, pharmacokinetics, and distribution.ResultsThe current research shows BM1 has a low in vivo toxicity profile, affinity for the renal system in rats, and good absorption, distribution, metabolism, and excretion (ADME). BM1 also has potent activity against azole-resistant fungal strains, including C. auris isolates and CDR6-overexpressing strains.ConclusionsThe results confirmed low minimum inhibitory concentrations (MICs) against several Candida species, including preliminary data vs. C. auris. BM1 has good ADME and biochemical characteristics, is suitable and safe for daily administration and is particularly indicated for renal infections. These data indicate BM1 and its derivatives form a novel, promising antifungal class.     

Antifungal Spectrum,In Vivo Efficacy,and Structure–Activity Relationship of Ilicicolin H

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A comprehensive review on potential applications of metallic nanoparticles as antifungal therapies to combat human fungal diseases

Human pathogenic fungi are responsible for causing a range of infection types including mucosal, skin, and invasive infections. Life-threatening and invasive fungal infections (FIs) are responsible for mortality and morbidity, especially for individuals with compromised immune function. The number of currently available therapeutic agents against invasive FIs is limited compared to that against bacterial infections. In addition, the increased mortality and morbidity caused by FIs are linked to the limited number of available antifungal agents, antifungal resistance, and the increased toxicity of these agents. Currently available antifungal agents have several drawbacks in efficiency, efficacy, toxicity, activity spectrum, and selectivity. It has already been demonstrated with numerous metallic nanoparticles (MNPs) that these nanoparticles can serve as an effective and alternative solution as fungicidal agents. MNPs have great potential owing to their intrinsic antifungal properties and potential to deliver antifungal drugs. For instance, gold nanoparticles (AuNPs) have the capacity to disturb mitochondrial calcium homeostasis induced AuNP-mediated cell death in Candida albicans. In addition, both copper nanoparticles and copper oxide nanoparticles exerted significant suppressive properties against pathogenic fungi. Silver nanoparticles showed strong antifungal properties against numerous pathogenic fungi, such as Stachybotrys chartarum, Mortierella alpina, Chaetomium globosum, A. fumigatus, Cladosporium cladosporioides, Penicillium brevicompactum, Trichophyton rubrum, C. tropicalis, and C. albicans. Iron oxide nanoparticles showed potent antifungal activities against A. niger and P. chrysogenum. It has also been reported that zinc oxide nanoparticles can significantly inhibit fungal growth. These NPs have already exerted potent antifungal properties against a number of pathogenic fungal species including Candida, Aspergillus, Fusarium, and many others. Several strategies are currently used for the research and development of antifungal NPs including chemical modification of NPs and combination with the available drugs. This review has comprehensively presented the current and innovative antifungal approach using MNPs. Moreover, different types of MNPs, their physicochemical characteristics, and production techniques have been summarized in this review.     

The effect of antifungal combination on transcripts of a subset of drug-resistance genes in clinical isolates of Candida species induced biofilms

Biofilm formation is often associated with increased Candida resistance toward antifungal agents. Therefore, the current study aimed to assess the incidence of biofilm formation among Candida isolates and to investigate the effect of high doses of fluconazole {FLC}, voriconazole {VOC} and amphotericin B {AMB}, singly and in combination on mature biofilms. Moreover, it aimed to assess the expression of selected genes (CDR1, KRE1 and SKN1) responsible for Candida biofilm resistance. The study included 49 patients; samples were collected from the King Khalid Hospital, Riyadh, Saudi Arabia. Isolates were prepared for biofilm formation and quantification using 0.4% (w/v) crystal violet. Minimum Inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) were conducted by the broth microdilution method. Biofilm eradication was evaluated using counting, XTT stain intensity and observed under the inverted microscope. Selected genes were evaluated in Candida biofilms under the effect of antifungal exposure using QPCR. The major isolates were Candida albicans (65.3%) followed by Candida tropicalis and Candida glabrata. 77.6% of the strains were biofilm formers. AMB showed susceptibility in 87.8% of isolates, followed by VOC (77.6%) and FLC (67.3%). MIC50 and MIC90 were (0.03, 0.125), (0.5, 8), (2, >128) μg/ml for AMB, VOC and FLC, respectively. 34.7% and 18.4% of the isolates were antagonistic to AMB/FLC and AMB/VOC, respectively. Mature biofilms of ten selected isolates were found resistant to FLC (1000 μg/ml). VOR and AMB concentration required to inhibit biofilm formation was 16–250 fold higher than the MIC for planktonic cells. Isolates showed significant reduction with antifungal combination when compared with the untreated controls (p value ⩽ 0.01), or using fluconazole alone (p value ⩽ 0.05). High doses of the antifungals were employed to assess the effect on the persisters’ selected gene expression. Marked over expression of SKN1 and to a lesser extent KRE1 was noticed among the mature biofilms treated with AMB alone or in combination after 1 h of exposure, and SKN1 expression was even more sharply induced after 24 h. No statistically significant over expression of CDR1 was observed in biofilms after exposure to high doses of FLC, VOC or any of the combinations used.     

Fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole

Silver nanoparticles (Ag-NPs) are known to have inhibitory and bactericidal effects. Resistance of fungal infections has emerged in recent years and is a major health problem. Here, we report the extracellular biosynthesis of Ag-NPs using a common fungus, Alternaria alternata. Also in this study, these nanoparticles were evaluated for their part in increasing the antifungal activity of fluconazole against Phoma glomerata, Phoma herbarum, Fusarium semitectum, Trichoderma sp., and Candida albicans. The antifungal activity of fluconazole was enhanced against the test fungi in the presence of Ag-NPs. Fluconazole in combination with Ag-NPs showed the maximum inhibition against C. albicans, which was confirmed from the increase in fold area of inhibition, followed by P. glomerata and Trichoderma sp., which showed less increase in the fold area, whereas no significant enhancement of activity was found against P. herbarum and F. semitectum.From the Clinical EditorThe antifungal activity of fluconazole was enhanced in presence of silver nanoparticles against the test fungi. Fluconazole in combination with Ag-NPs showed the maximum inhibition against C. albicans, followed by P. glomerata and Trichoderma sp. No significant enhancement of activity was found against P. herbarum and F. semitectum.     

Evaluation of a novel oxiconazole nitrate formulation: The thermosensitive gel

Superficial fungal infections caused by Candida species are common skin diseases. Therefore, this study aimed to develop a new formulation containing oxiconazole nitrate, which is an azole group derivative for antifungal treatment, as a thermosensitive gel since there has been no literature study until now.MIC value of the novel thermosensitive formulation against three Candida species was calculated and time-dependent antifungal activity analysis was performed. Viscosity, transition temperature T_sol-gel (°C) and gelation time of the thermosensitive gel formulation were also determined in the viscometer. The measurements performed on the tensilometer device were analyzed for adhesion hardness and elongation percentages of the formulation. In the FT-IR spectrometer, the spectrum of solution and gel state was compared between 650 and 4000?cm^?1 and it was found that there is no difference between them.It was found that the temperature is reversible on the formulation and did not cause any disruption of its components. Characterization parameters of the thermosensitive gel formulation containing oxiconazole nitrate and time-dependent activity against Candida species was observed to be the same as those of the solution containing only oxiconazole nitrate. MIC, MFC and time-dependent antifungal analysis did not show any particular difference between formulation and oxiconazole nitrate itself. Thermosensitive gel formulation containing oxiconazole nitrate was found to be effective on superficial fungal infections. We believe it is also appropriate for in vivo usage, but it is necessary to perform animal and human research. It is also needed to evaluate the formulation against other etiologic agents of superficial fungal infections.