New Antifungal Agents and New Formulations Against Dermatophytes

A variety of oral and topical antifungal agents are available for the treatment of superficial fungal infections caused by dermatophytes. This review builds on the antifungal therapy update published in this journal for the first special issue on Dermatophytosis (Gupta and Cooper 2008;166:353–67). Since 2008, there have not been additions to the oral antifungal armamentarium, with terbinafine, itraconazole, and fluconazole still in widespread use, albeit for generally more severe or recalcitrant infections. Griseofulvin is used in the treatment of tinea capitis. Oral ketoconazole has fallen out of favor in many jurisdictions due to risks of hepatotoxicity. Topical antifungals, applied once or twice daily, are the primary treatment for tinea pedis, tinea corporis/tinea cruris, and mild cases of tinea unguium. Newer topical antifungal agents introduced include the azoles, efinaconazole, luliconazole, and sertaconazole, and the oxaborole, tavaborole. Research is focused on developing formulations of existing topical antifungals that utilize novel delivery systems in order to enhance treatment efficacy and compliance.     

Relevant Animal Models in Dermatophyte Research

Dermatophytoses are common superficial fungal infections affecting both humans and animals. They are provoked by filamentous fungi called dermatophytes specialized in the degradation of keratinized structures, which allows them to induce skin, hair and nail infections. Despite their high incidence, little investigation has been performed for the understanding of these infections compared to fungal opportunistic infections and most of the studies were based on in vitro experiments. The development of animal models for dermatophyte research is required to evaluate new treatments against dermatophytoses or to increase knowledge about fungal pathogenicity factors or host immune response mechanisms. The guinea pig has been the most often used animal model to evaluate efficacy of antifungal compounds against dermatophytes, while mouse models were preferred to study the immune response generated during the disease. Here, we review the relevant animal models that were developed for dermatophyte research and we discuss the advantages and disadvantages of the selected species, especially guinea pig and mouse.     

Rare Fungal Infections in Children: An Updated Review of the Literature

An increasing trend of reports of rare fungal diseases has been observed to be mainly associated with the substantial increase of high-risk immunocompromised children, as well as with the selective pressure of antifungal drugs. On the other hand, recent reports have shown that several species of these rare fungi may also cause infections in immunocompetent children without obvious underlying conditions. The clinical spectrum of these infections, and most importantly their outcome, varies greatly, implying for a rather heterogenic group of pediatric infections. Various types of superficial and subcutaneous fungal infections, as well as systemic and disseminated life-threatening infections, have been reported. Prompt diagnosis and appropriate treatment of rare fungal diseases in children remains a great challenge. Several treatment options have been used, ranging from localized to combination treatment with extensive surgical excision and long-term antifungal therapy. We review contemporary data of rare fungal infections in pediatric patients focusing on epidemiology, mycology, management and outcome, published during the last three years.     

Biomaterials surfaces capable of resisting fungal attachment and biofilm formation

Microbial attachment onto biomedical devices and implants leads to biofilm formation and infection; such biofilms can be bacterial, fungal, or mixed. In the past 15 years, there has been an increasing research effort into antimicrobial surfaces but the great majority of these publications present research on bacteria, with some reports also testing resistance to fungi. Very few studies have focused exclusively on antifungal surfaces. However, with increasing recognition of the importance of fungal infections to human health, particularly related to infections at biomaterials, it would seem that the interest in antifungal surfaces is disproportionately low. In studies of both bacteria and fungi, fungi tend to be the minor focus with hypothesized antibacterial mechanisms of action often generalized to also explain the antifungal effect. Yet bacteria and fungi represent two Distinct biological Domains and possess substantially different cellular physiology and structure. Thus it is questionable whether these generalizations are valid. Here we review the scientific literature focusing on surface coatings prepared with antifungal agents covalently attached to the biomaterial surface. We present a critical analysis of generalizations and their evidence. This review should be of interest to researchers of “antimicrobial” surfaces by addressing specific issues that are key to designing and understanding antifungal biomaterials surfaces and their putative mechanisms of action.     

A Prodrug Approach to the Use of Coumarins as Potential Therapeutics for Superficial Mycoses

Superficial mycoses are fungal infections of the outer layers of the skin, hair and nails that affect 20–25% of the world''s population, with increasing incidence. Treatment of superficial mycoses, predominantly caused by dermatophytes, is by topical and/or oral regimens. New therapeutic options with improved efficacy and/or safety profiles are desirable. There is renewed interest in natural product-based antimicrobials as alternatives to conventional treatments, including the treatment of superficial mycoses. We investigated the potential of coumarins as dermatophyte-specific antifungal agents and describe for the first time their potential utility as topical antifungals for superficial mycoses using a prodrug approach. Here we demonstrate that an inactive coumarin glycone, esculin, is hydrolysed to the antifungal coumarin aglycone, esculetin by dermatophytes. Esculin is hydrolysed to esculetin β-glucosidases. We demonstrate that β-glucosidases are produced by dermatophytes as well as members of the dermal microbiota, and that this activity is sufficient to hydrolyse esculin to esculetin with concomitant antifungal activity. A β-glucosidase inhibitor (conduritol B epoxide), inhibited antifungal activity by preventing esculin hydrolysis. Esculin demonstrates good aqueous solubility (<6 g/l) and could be readily formulated and delivered topically as an inactive prodrug in a water-based gel or cream. This work demonstrates proof-of-principle for a therapeutic application of glycosylated coumarins as inactive prodrugs that could be converted to an active antifungal in situ. It is anticipated that this approach will be applicable to other coumarin glycones.     

Photodynamic Antifungal Therapy Against Chromoblastomycosis

Photodynamic therapy (PDT) is a minimally invasive approach, in which a photosensitizer compound is activated by exposure to light. The activation of the sensitizer drug results in several chemical reactions, such as the production of reactive oxygen species and other reactive molecules, which presence in the biological site leads to the damage of target cells. Although PDT has been primarily developed to combat cancerous lesions, this therapy can be employed for the treatment of several conditions, including infectious diseases. A wide range of microorganisms, including Gram-positive and Gram-negative bacteria, viruses, protozoa, and fungi, have demonstrated susceptibility to antimicrobial PDT. This treatment might consist in an alternative for the management of fungal infections. Antifungal photodynamic therapy has been successfully employed against Candida species, dermatophytes, and Aspergillus niger. Chromoblastomycosis is an infection that involves skin and subcutaneous tissues caused by the traumatic inoculation of dematiaceous fungi species, being that the most prevalent are Fonsecaea pedrosoi and Claphialophora carrionii. In the present work, the clinical applications of PDT for the treatment of chromoblastomycosis are evaluated. We have employed methylene blue as photosensitizer and a LED (Light Emitting Diode) device as light source. The results of this treatment are positive, denoting the efficacy of PDT against chromoblastomycosis. Considering that great part of the published works are focused on in vitro trials, these clinical tests can be considered a relevant source of information about antifungal PDT, since its results have demonstrated to be promising. The perspectives of this kind of treatment are analyzed in agreement with the recent literature involving antifungal PDT.     

Major challenges and perspectives in the diagnostics and treatment of dermatophyte infections

Dermatophytes are the aetiological factors of a majority of superficial fungal infections. What distinguishes them from other pathogenic filamentous fungi is their unique ability to degrade keratin. The remarkable ability of this group of fungi to survive in different ecosystems results from their morphological and ecological diversity as well as high adaptability to changing environmental conditions. Paradoxically, despite the progress in medicine, the prevalence of dermatophyte infections is increasing from year to year. At the beginning of the third millennium, practical diagnostic and therapeutic options are still very limited. This review focuses on understanding the major problems in this aspect of dermatophyte infections and indicates future strategies and perspectives for novel approaches to identification and drugs for elimination of dermatophytes. Particular importance is placed on development of a strategy for a diagnostic pathway and implementation of rapid and reliable diagnostics methods designed by international teams. Furthermore, among compounds that currently arouse great interest, representatives of terpenoids, alkaloids, saponins, flavonoids and essential oils deserve attention. Many of these compounds are undergoing clinical trials as potential antifungal agents, and future research should focus on attempts at determination of the applicability of tested substances. Finally, the advantages and disadvantages in implementation of new diagnostic paths and medicinal substances for routine use are indicated.     

Sequenced dermatophyte strains: growth rate, conidiation, drug susceptibilities, and virulence in an invertebrate model

Although dermatophytes are the most common cause of fungal infections in the world, their basic biology is not well understood. The recent sequencing and annotation of the genomes of five representative dermatophyte species allows for the creation of hypotheses as to how they cause disease and have adapted to their distinct environments. An understanding of the microbiology of these strains will be essential for testing these hypotheses. This study is the first to generally characterize these five sequenced strains of dermatophytes for their microbiological aspects. We measured the growth rate on solid medium and found differences between species, with Microsporum gypseum CBS118893 having the fastest growth and Trichophyton rubrum CBS118892 the slowest. We also compared different media for conidia production and found that the highest numbers of conidia were produced when dermatophytes were grown on MAT agar. We determined the Minimum Inhibitory Concentration (MIC) of nine antifungal agents and confirmed susceptibility to antifungals commonly used as selectable markers. Finally, we tested virulence in the Galleria mellonella (wax moth) larvae model but found the results variable. These results increase our understanding of the microbiology and molecular biology of these dermatophyte strains and will be of use in advancing hypothesis-driven research about dermatophytes.     

Invasive Pulmonary Fungal Infections in Cystic Fibrosis

Invasive pulmonary mycosis is after allergic bronchopulmonary aspergillosis (ABPA) a frequent and severe complication of CF lung disease. Among CF caregivers, there is an insecurity when and how to treat infections of the lung parenchyma caused by different fungi in patients with CF. This case series provides a multicenter experience on diagnostic, manifestation, and treatment of non-ABPA cases of pulmonary. Non-ABPA cases of pulmonary mycoses in patients with CF have been collected from the CF Centers in Berlin, Essen, Worms, Frankfurt (Germany), Leeds (UK), and Barcelona (Spain). Non-ABPA was defined as total serum IgE level <500 kU/L. Scedosporium and Lomentospora species seem to be more virulent in patients with CF and have been successfully treated with triple antifungal drug regimens in several cases. Rare fungi including yeasts can have pathogenic potential in CF. In this series, antibiotic treatment failure was the main indicator for the initiation of antifungal treatment. For an early and effective treatment of pulmonary mycoses in CF, the identification of biomarkers and of risk factors beyond antibiotic treatment failure is crucial and urgently needed. Furthermore, treatment efficacy studies are necessary for the different causative agents of these infections.     

Updating Corneofungimetry: A Bioassay Exploring Dermatomycoses and Antifungal Susceptibility

Superficial dermatomycoses are frequent conditions in humans and animals. Specific treatment modalities have been designed using a variety of different antifungal compounds. The need for antifungal susceptibility testing (AST) has been growing steadily over the last two decades due to the extending number of newer antifungal agents. Objective inter- and intraindividual comparisons of their respective efficacies are nearly impossible to perform in vivo. Currently, a series of standardized AST methods and interpretative guidelines have been designed. However, their clinical relevance for dermatomycoses is not consistent. The corneofungimetry bioassay was designed to test comparatively a series of antifungals on pathogenic fungi growing on sheets of human stratum corneum. Computerized morphometric assessments bring numerical values allowing statistical comparisons. Variants of corneofungimetry address more specific aspects related to fungal cell adhesion, fungitoxicity and lipid-dependent fungi.     

Haloprogin: a Topical Antifungal Agent

Haloprogin was shown to be a highly effective agent for the treatment of experimentally induced topical mycotic infections in guinea pigs. Its in vitro spectrum of activity also includes yeasts, yeastlike fungi (Candida species), and certain gram-positive bacteria. The in vitro and in vivo antifungal activity of haloprogin against dermatophytes was equal to that observed with tolnaftate. The striking differences between the two agents were the marked antimonilial and selective antibacterial activities shown by haloprogin, contrasted with the negligible activities found with tolnaftate. Addition of serum decreased the in vitro antifungal activity of haloprogin to a greater extent than that of tolnaftate; however, diminished antifungal activity was not observed when haloprogin was applied topically to experimental dermatophytic infections. Based on its broad spectrum of antimicrobial activity, haloprogin may prove to be a superior topical agent in the treatment of dermatophytic and monilial infections in man.     

Update on the diagnosis of dermatomycosis

Dermatomycosis are mycotic diseases of skin caused by a few mycetes: dermatophytes, and some opportunistic fungi as Malassezia, Candida (not C. albicans), Trichosporon, Rhodutorula, Cryptococcus or Aspergillus, Geotrichum, Alternaria, etc. Dermatophytes are a group of closely related filamentous fungi that invade keratinized tissue (skin, hair, nails) of humans and other animals and produce infection called dermatophytosis or ringworm or "tinea". The etiological agents of dermatophytosis are classified in three genera: Microsporum, Trichophyton and Epidermophyton (Deuteromycetes). On the basis of their primary habitat dermatophytes are divided in Anthropophilic dermatophytes (parasitic organisms that infect humans), Zoophilic dermatophytes (parasitic organisms that infect animals, but also humans: agents of zoonosis) and Geophilic dermatophytes (saprobic fungi associated with keratinous materials in soil). In the soil there are also structure associated with contagion, ("spore", "arthroconidium", or "clamydospore") of anthropophilic and zoophilic dermatophytes that may persist for years, in the environment, in hair or skin scales. Since on the skin of animals there are many saprobic organisms (Malassezia) and many fungi may infect the fur, it is important to make an accurate diagnosis. Dermatophytosis are communicable diseases acquired from infected animals or from fomites. Infections caused by dermatophytes is a ringworm. These infections may range from mild and superficial, almost subclinical, to a few areas of scaling to a highly inflammatory reaction with extensive areas of scarring and alopecia. Granuloma formations (mycetoma-like) may occur especially in cats. Dermatophytes, as filamentous fungi, undergo radial fungi: collection of skin material is best made by collecting the scales near the edges of the rings. Hairs are best sampled by plucking; a scalpel may be used to scrape scales; brushes have also been used. Sample materials are best transported in dry packet. The Wood's light may be used to identify infected fluorescent hairs. Direct microscopy, although false negative up to 50% of cases, is a highly efficient screening technique. Scraping and hairs should mixed to 10-15% KOH. Culture is a valuable adjunct to direct microscopy and is essential to identify more dermatophytes. A medium selective against most nondermatophytic moulds and bacteria is used as a primary isolation medium. Many typical isolates of common dermatophytes can be identified directly from primary isolation media. Identification characters include: colony pigmentation, texture, morphological structure (macroconidia, microconidia, spirals, pectinate branches, etc). Nutritional requiment, growth in special media, "in vitro" perforation, mating studies are procedures used to identify atypical isolates. Serological approaches have revealed difficulties. Many kinds of molecular biologic techniques have been made available for clinical diagnosis recently; almost all of these techniques involve the polymerase chain reaction (PCR).     

Photodynamic inactivation for controlling Candida albicans infections

Antimicrobial photodynamic therapy (APDT) combines a non-toxic dye, termed photosensitizer, which is activated by visible light of appropriate wavelength which will produce reactive oxygen species (ROS). These ROS will react with cellular components inducing oxidative processes, leading to cell death. A wide range of microorganisms, have already showed susceptibility to APDT. Therefore, this treatment might consist in an alternative for the management of fungal infections that is mainly caused by biofilms, since they respond poorly to conventional antibiotics and may play a role in persistent infections. Biofilms are the leading cause of microbial infections in humans, thus representing a serious problem in health care. Candida albicans is the main type of fungi able to form biofilms, which cause superficial skin and mucous membrane infections as well as deep-seated mycoses, particularly in immunocompromised patients. In these patients, invasive infections are often associated with high morbidity and mortality. Furthermore, the increase in antifungal resistance has decreased the efficacy of conventional therapies. Treatments are time-consuming and thus demanding on health care budgets. Additionally, current antifungal drugs only have a limited spectrum of action and toxicity. The use of APDT as an antimicrobial topical agent against superficial and cutaneous diseases represents an effective method for eliminating microorganisms.     

The fungal cell wall as a target for the development of new antifungal therapies

In the past three decades invasive mycoses have globally emerged as a persistent source of healthcare-associated infections. The cell wall surrounding the fungal cell opposes the turgor pressure that otherwise could produce cell lysis. Thus, the cell wall is essential for maintaining fungal cell shape and integrity. Given that this structure is absent in host mammalian cells, it stands as an important target when developing selective compounds for the treatment of fungal infections. Consequently, treatment with echinocandins, a family of antifungal agents that specifically inhibits the biosynthesis of cell wall (1-3)β-D-glucan, has been established as an alternative and effective antifungal therapy. However, the existence of many pathogenic fungi resistant to single or multiple antifungal families, together with the limited arsenal of available antifungal compounds, critically affects the effectiveness of treatments against these life-threatening infections. Thus, new antifungal therapies are required. Here we review the fungal cell wall and its relevance in biotechnology as a target for the development of new antifungal compounds, disclosing the most promising cell wall inhibitors that are currently in experimental or clinical development for the treatment of some invasive mycoses.     

Susceptibility of clinical isolates of fungi to saperconazole

The in vitro activity of saperconazole against 228 strains of mycotic agents belonging to 48 species was investigated. Susceptibility testing was performed using a microtiter broth dilution method. Isolates of Candida albicans, C. tropicalis and Torulopsis glabrata showed distinct intra-species variation of susceptibility with MIC values ranging from 0.045 to 100 mg l^–1. The drug was inhibitory for the dermatophytes at a relatively narrow range of concentrations, most isolates being inhibited at MIC 0.78 mg l^–1. The strongest antifungal potency of saperconazole was exerted against clinical isolates of the genus Aspergillus (MIC 90% = 0.19 mg l^–1). Concentrations up to 100 mg l^–1 had no macroscopically recognizable effect on the growth of zygomycetous fungi (Mucor, Rhizopus, Syncephalastrum). Species of the genus Absidia with their good sensitivity are an exception. Justification of in vitro susceptibility testing of triazoles is discussed. In the author's opinion, MIC values can serve as an informative parameter showing the range of indications of these antifungals for treatment. It is concluded that saperconazole exhibits a very good activity against a broad spectrum of medically important fungi in vitro and can be considered a promising antifungal drug.     

Phenotypic characterization of Aspergillus niger and Candida albicans grown under simulated microgravity using a three-dimensional clinostat

The living and working environments of spacecraft become progressively contaminated by a number of microorganisms. A large number of microorganisms, including pathogenic microorganisms, some of which are fungi, have been found in the cabins of space stations. However, it is not known how the characteristics of microorganisms change in the space environment. To predict how a microgravity environment might affect fungi, and thus how their characteristics could change on board spacecraft, strains of the pathogenic fungi Aspergillus niger and Candida albicans were subjected to on-ground tests in a simulated microgravity environment produced by a three-dimensional (3D) clinostat. These fungi were incubated and cultured in a 3D clinostat in a simulated microgravity environment. No positive or negative differences in morphology, asexual reproductive capability, or susceptibility to antifungal agents were observed in cultures grown under simulated microgravity compared to those grown in normal earth gravity (1 G). These results strongly suggest that a microgravity environment, such as that on board spacecraft, allows growth of potentially pathogenic fungi that can contaminate the living environment for astronauts in spacecraft in the same way as they contaminate residential areas on earth. They also suggest that these organisms pose a similar risk of opportunistic infections or allergies in astronauts as they do in people with compromised immunity on the ground and that treatment of fungal infections in space could be the same as on earth.     

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

Invasive Aspergillosis: Resistance to Antifungal Drugs

Although the arsenal of agents with anti-Aspergillus activity has expanded over the last decade, mortality due to invasive aspergillosis remains unacceptably high. Resistance of the Aspergillus spp. species to antifungal drugs increased in the last 20 years with the increase in antifungal drugs use and might partially account for treatment failures. Recent advances in our understanding of mechanisms of antifungal drug action in Aspergillus, along with the standardization of in vitro susceptibility testing methods, have brought resistance testing to the forefront of clinical mycology. Recent modifications in taxonomy and understanding of the acquired resistance mechanisms of Aspergilli to drugs should support a better management of Aspergillus infections. In this paper, we review the current knowledge on epidemiology and underlying mechanisms involved in antifungal resistance in Aspergillus.     

Antifungal susceptibility testing: Recent findings and experience

The current status of antifungal susceptibility tests is very much in flux. There is ample evidence that current testing of fungi is at this point poorly standardized, and therefore the results from such tests are difficult to apply to patient management. On the other hand, there is now considerable interest in collaborative efforts to change this situation and all evidence to date indicates that standardization is a reasonable and clearly achievable goal. In the NCCLS Subcommittee Report, ²⁹ several steps were proposed to reach that objective. Among others, they include: 1) Establishing a panel of fungal isolates to be used in further studies; 2) Establish performance standards for a single media that might be used to test all known classes of antifungal agents; and 3) Determine and correct the sources of error that result in variability of the broth dilution method.Now that the problem has been more sharply defined, work to eliminate interlaboratory variability and to determine test correlation with treatment results should proceed. Hopefully work during the next few years will result in antifungal tests that are just as useful as today's tests for antibacterial agents.