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Acylhydrazone (AH) derivatives represent a novel category of anti-fungal medications that exhibit potent activity against Sporothrix sp., both in vitro and in a murine model of sporotrichosis. In this study, we demonstrated the anti-fungal efficacy of the AH derivative D13 [4-bromo- N '-(3,5-dibromo-2-hydroxybenzylidene)-benzohydrazide] against both planktonic cells and biofilms formed by Sporothrix brasiliensis . In a clinical study, the effect of D13 was then tested in combination with itraconazole (ITC), with or without potassium iodide, in 10 cats with sporotrichosis refractory to the treatment of standard of care with ITC. Improvement or total clinical cure was achieved in five cases after 12 weeks of treatment. Minimal abnormal laboratory findings, e.g., elevation of alanine aminotransferase, were observed in four cats during the combination treatment and returned to normal level within a week after the treatment was ended. Although highly encouraging, a larger and randomized controlled study is required to evaluate the effectiveness and the safety of this new and exciting drug combination using ITC and D13 for the treatment of feline sporotrichosis., Importance: This paper reports the first veterinary clinical study of an acylhydrazone anti-fungal (D13) combined with itraconazole against a dimorphic fungal infection, sporotrichosis, which is highly endemic in South America in animals and humans. Overall, the results show that the combination treatment was efficacious in ~50% of the infected animals. In addition, D13 was well tolerated during the course of the study. Thus, these results warrant the continuation of the research and development of this new class of anti-fungals., Competing Interests: M.D.P. is a co-founder and CSO; J.B.M. is a co-founder and CEO; and J.M. is a co-founder and chief research and development officer of MicroRid Technologies Inc. The goal of MicroRid Technologies Inc. is to develop new anti-fungal agents of therapeutic use. All other authors declare no competing interests.

Mucorales are a group of non-septated filamentous fungi widely distributed in nature, frequently associated with human infections, and are intrinsically resistant to many antifungal drugs. For these reasons, there is an urgent need to improve the clinical management of mucormycosis. Miltefosine, which is a phospholipid analogue of alkylphosphocholine, has been considered a promising repurposing drug to be used to treat fungal infections. In the present study, miltefosine displayed antifungal activity against a variety of Mucorales species, and it was also active against biofilms formed by these fungi. Treatment with miltefosine revealed modifications of cell wall components, neutral lipids, mitochondrial membrane potential, cell morphology, and the induction of oxidative stress. Treated Mucorales cells also presented an increased susceptibility to SDS. Purified ergosterol and glucosylceramide added to the culture medium increased miltefosine MIC, suggesting its interaction with fungal lipids. These data contribute to elucidating the effect of a promising drug repurposed to act against some relevant fungal pathogens that significantly impact public health.

Candida species are one of the most concerning causative agents of fungal infections in humans. The treatment of invasive Candida infections is based on the use of fluconazole, but the emergence of resistant isolates has been an increasing concern which has led to the study of alternative drugs with antifungal activity. Sphingolipids have been considered a promising target due to their roles in fungal growth and virulence. Inhibitors of the sphingolipid biosynthetic pathway have been described to display antifungal properties, such as myriocin and aureobasidin A, which are active against resistant Candida isolates. In the present study, aureobasidin A did not display antibiofilm activity nor synergism with amphotericin B, but its combination with fluconazole was effective against Candida biofilms and protected the host in an in vivo infection model. Alterations in treated cells revealed increased oxidative stress, reduced mitochondrial membrane potential and chitin content, as well as altered morphology, enhanced DNA leakage and a greater susceptibility to sodium dodecyl sulphate (SDS). In addition, it seems to inhibit the efflux pump CaCdr2p. All these data contribute to elucidating the role of aureobasidin A on fungal cells, especially evidencing its promising use in clinical resistant isolates of Candida species.

Sporothrix brasiliensis is the main agent of zoonotic sporotrichosis transmitted by domestic cats in South America. In humans, sporotrichosis commonly presents with cutaneous or lymphocutaneous lesions, and in cats, with multiple ulcerated skin lesions associated with enlarged lymph nodes and respiratory signs. Fungal virulence factors may affect the clinical presentation of the mycoses. Sporothrix spp. present some virulence factors. This study aims to compare 24 S. brasiliensis strains from 12 familiar outbreaks of cat-to-human transmitted sporotrichosis. Fungal growth in different substrates, thermotolerance, resistance to oxidative stress, and production of enzymes were evaluated. An invertebrate model of experimental infection was used to compare the virulence of the strains. The strains grew well on glucose and N -acetyl- D -glucosamine but poorly on lactate. Their thermotolerance was moderate to high. All strains were susceptible to hydrogen peroxide, and the majority produced hemolysins but not phospholipase and esterase. There was no significant difference in the putative virulence-associated factors studied among the different hosts. Moreover, strains isolated from a human and a cat from four familiar outbreaks presented a very similar profile of expression of these factors, reinforcing the zoonotic transmission of S. brasiliensis in Brazil and demonstrating the plasticity of this species in the production of virulence factors.

Scedosporium and Lomentospora species are opportunistic filamentous fungi that cause localized and disseminated infections in immunocompetent and immunocompromised patients. These species are considered resistant fungi due to their low susceptibility to most current antifungal agents used in healthcare settings. The search for new compounds that could work as promising candidate antifungal drugs is an increasing field of interest. In this context, in the present study we screened the Pandemic Response Box® library (Medicines for Malaria Venture [MMV], Switzerland) to identify compounds with antifungal activity against Scedosporium and Lomentospora species. An initial screening of the drugs from this collection at 5 μM was performed using a clinical Scedosporium aurantiacum isolate according to the EUCAST protocol. Compounds with activity against this fungus were also tested against four other species (S. boydii¸ S. dehoogii, S. apiospermum and L. prolificans) at concentrations ranging from 0.078 to 10 μM. Seven compounds inhibited more than 80% of S. aurantiacum growth, three of them (alexidine, amorolfine and olorofim) were selected due to their differences in mechanism of action, especially when compared to drugs from the azole class. These compounds were more active against biofilm formation than against preformed biofilm in Scedosporium and Lomentospora species, except alexidine, which was able to decrease preformed biofilm about 50%. Analysis of the potential synergism of these compounds with voriconazole and caspofungin was performed by the checkerboard method for S. aurantiacum. The analysis by Bliss methodology revealed synergistic effects among selected drugs with caspofungin. When these drugs were combined with voriconazole, only alexidine and amorolfine showed a synergistic effect, whereas olorofim showed an antagonistic effect. Scanning electron microscopy revealed that alexidine induces morphology alterations in S. aurantiacum biofilm grown on a catheter surface. Reactive oxygen species production, mitochondrial activity and surface components were analyzed by fluorescent probes when S. aurantiacum was treated with selected drugs and revealed that some cell parameters are altered by these compounds. In conclusion, alexidine, amorolfine and olorofim were identified as promising compounds to be studied against scedosporiosis and lomentosporiosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rollin-Pinheiro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mucormycosis is considered concerning invasive fungal infections due to its high mortality rates, difficult diagnosis and limited treatment approaches. Mucorales species are highly resistant to many antifungal agents and the search for alternatives is an urgent need. In the present study, a library with 400 compounds called the Pandemic Response Box ® was used and four compounds were identified: alexidine and three non-commercial molecules. These compounds showed anti-biofilm activity, as well as alterations in fungal morphology and cell wall and plasma membrane structure. They also induced oxidative stress and mitochondrial membrane depolarization. In silico analysis revealed promising pharmacological parameters. These results suggest that these four compounds are potent candidates to be considered in future studies for the development of new approaches to treat mucormycosis.

The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the Sporothrix pathogenic species. The initial screen identified twenty-four candidates that inhibited the growth of Sporothrix brasiliensis by more than 80%. Some of these compounds are known to display antifungal activity, including olorofim (MMV1782354), a new antifungal drug. Olorofim inhibited and killed the yeasts of S. brasiliensis , S. schenckii , and S. globosa at concentrations lower than itraconazole, and it also showed antibiofilm activity. According to the results obtained by fluorimetry, electron microscopy, and particle characterization analyses, we observed that olorofim induced profound alterations on the cell surface and cell cycle arrest in S. brasiliensis yeasts. We also verified that these morphophysiological alterations impaired their ability to adhere to keratinocytes in vitro. Our results indicate that olorofim is a promising new antifungal against sporotrichosis agents.

Sporotrichosis, a human and animal disease caused by Sporothrix species, is the most important implantation mycosis worldwide. Sporothrix taxonomy has improved in recent years, allowing important advances in diagnosis, epidemiology, and treatment. Molecular epidemiology reveals that S. brasiliensis remains highly prevalent during the cat-transmitted sporotrichosis outbreaks in South America and that the spread of S. brasiliensis occurs through founder effects. Sporothrix globosa and S. schenckii are cosmopolitan on the move, causing major sapronoses in Asia and the Americas, respectively. In this emerging scenario, one-health approaches are required to develop a creative, effective, and sustainable response to tackle the spread of sporotrichosis. In the 21st century, it has become vital to speciate Sporothrix , and PCR is the main pillar of molecular diagnosis, aiming at the detection of the pathogen DNA from clinical samples through multiplex assays, whose sensitivity reaches remarkably three copies of the target. The treatment of sporotrichosis can be challenging, especially after the emergence of resistance to azoles and polyenes. Alternative drugs arising from discoveries or repositioning have entered the radar of basic research over the last decade and point to several molecules with antifungal potential, especially the hydrazone derivatives with great in vitro and in vivo activities. There are many promising developments for the near future, and in this review, we discuss how these trends can be applied to the Sporothrix -sporotrichosis system to mitigate the advance of an emerging and re-emerging disease., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Sporotrichosis is a subcutaneous mycosis that affects humans and animals, with few therapeutic options available in the pharmaceutical market. We screened the in vitro antifungal activity of fourteen 1,4-naphthoquinones derivative compounds against Sporothrix brasiliensis and Sporothrix schenckii, the main etiological agents of sporotrichosis in Latin America. The most active compound was selected for further studies exploring its antibiofilm activity, effects on yeast morphophysiology, interaction with itraconazole, and selectivity to fungal cells. Among the fourteen 1,4-naphthoquinones tested, naphthoquinone 5, a silver salt of lawsone, was the most active compound. Naphthoquinone 5 was able to inhibit Sporothrix biofilms and induced ROS accumulation, mitochondrial disturbances, and severe plasmatic membrane damage in fungal cells. Furthermore, naphthoquinone 5 was ten times more selective towards fungal cells than fibroblast, and the combination of itraconazole with naphthoquinone 5 improved the inhibitory activity of the azole. Combined, the data presented here indicate that the silver salt naphthoquinone 5 exerts promising in vitro activity against the two main agents of sporotrichosis with important antibiofilm activity and a good toxicity profile, suggesting it is a promising molecule for the development of a new family of antifungals., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Sporotrichosis is the most prevalent subcutaneous mycosis globally, and it is typically caused by direct inoculation of the soil saprophytic fungus Sporothrix spp. into the patients' skin. However, sporotrichosis has an important zoonotic transmission route between cats and humans in hot-spot endemic areas such as Brazil. Antifungal itraconazole is the first-line treatment; however, it is frequently associated with recurrence after withdrawal, mainly on cats. Biofilms are important resistance structures related to the environmental persistence of most microorganisms. In the present work, we evaluated Sporothrix yeasts' ability to form biofilms in an ex vivo model of infected claws of cats. Using scanning electron microscopy, we demonstrated the presence of fungal biofilms in the claws of cats diagnosed with sporotrichosis confirmed by isolation of Sporothrix spp. in culture. We present here evidence of antibiofilm activity of miltefosine and suggest its use off-label as an antifungal as a putative alternative to itraconazole against Sporothrix biofilms. Claw contamination could sustain infections through a continuous inoculation cycle between open lesions and cat claws. Our results further support the off-label use of miltefosine as a promising alternative, especially for mycosis refractory to conventional treatment.

Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp . The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Ps d2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Ps d2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Ps d2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 μM Ps d2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Ps d2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Ps d2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Corrêa-Almeida, Borba-Santos, Rollin-Pinheiro, Barreto-Bergter, Rozental and Kurtenbach.)

Fungal infections have been increasing during the last decades. Scedosporium and Lomentospora species are filamentous fungi most associated to those infections, especially in immunocompromised patients. Considering the limited options of treatment and the emergence of resistant isolates, an increasing concern motivates the development of new therapeutic alternatives. In this context, the present study screened the Pathogen Box library to identify compounds with antifungal activity against Scedosporium and Lomentospora . Using antifungal susceptibility tests, biofilm analysis, scanning electron microscopy (SEM), and synergism assay, auranofin and iodoquinol were found to present promising repurposing applications. Both compounds were active against different Scedosporium and Lomentospora , including planktonic cells and biofilm. SEM revealed morphological alterations and synergism analysis showed that both drugs present positive interactions with voriconazole, fluconazole, and caspofungin. These data suggest that auranofin and iodoquinol are promising compounds to be studied as repurposing approaches against scedosporiosis and lomentosporiosis.

Sporotrichosis has become an important zoonosis in Brazil, and Sporothrix brasiliensis is the primary species transmitted by cats. Improvement of animal treatment will help control and limit the spread and geographic expansion of sporotrichosis. Accordingly, buparvaquone, an antiprotozoal hydroxynaphthoquinone agent marketed as Butalex, was evaluated in vitro and in vivo against feline-borne isolates of S. brasiliensis . Buparvaquone inhibited in vitro fungal growth at concentrations 4-fold lower than itraconazole (the first-choice antifungal used for sporotrichosis) and was 408 times more selective for S. brasiliensis than mammalian cells. Yeasts treated with a subinhibitory concentration of buparvaquone exhibited mitochondrial dysfunction, reactive oxygen species and neutral lipid accumulation, and impaired plasma membranes. Scanning electron microscopy images also revealed buparvaquone altered cell wall integrity and induced cell disruption. In vivo experiments in a Galleria mellonella model revealed that buparvaquone (single dose of 5 mg/kg of body weight) is more effective than itraconazole against infections with S. brasiliensis yeasts. Combined, our results indicate that buparvaquone has a great in vitro and in vivo antifungal activity against S. brasiliensis , revealing the potential application of this drug as an alternative treatment for feline sporotrichosis.

Scedosporium and Lomentospora species are filamentous fungi responsible for a wide range of infections in humans and are frequently associated with cystic fibrosis and immunocompromising conditions. Because they are usually resistant to many antifungal drugs available in clinical settings, studies of alternative targets in fungal cells and therapeutic approaches are necessary. In the present work, we evaluated the in vitro antifungal activity of miltefosine against Scedosporium and Lomentospora species and how this phospholipid analogue affects the fungal cell. Miltefosine inhibited different Scedosporium and Lomentospora species at 2-4 µg/ml and reduced biofilm formation. The loss of membrane integrity in Scedosporium aurantiacum caused by miltefosine was demonstrated by leakage of intracellular components and lipid raft disorganisation. The exogenous addition of glucosylceramide decreased the inhibitory activity of miltefosine. Reactive oxygen species production and mitochondrial activity were also affected by miltefosine, as well as the susceptibility to fluconazole, caspofungin and myoricin. The data obtained in the present study contribute to clarify the dynamics of the interaction between miltefosine and Scedosporium and Lomentospora cells, highlighting its potential use as new antifungal drug in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rollin-Pinheiro, Almeida, Rochetti, Xisto, Borba-Santos, Rozental and Barreto-Bergter.)

The in vitro activity of ibuprofen, a nonsteroidal anti-inflammatory drug, was evaluated against Sporothrix brasiliensis and S. schenckii, either alone or in combination with amphotericin B, itraconazole, or terbinafine. The inhibitory activity of ibuprofen as a single agent was determined according to minimum inhibitory concentration (MIC) values, while the effect of ibuprofen combined with amphotericin B, itraconazole, or terbinafine was estimated by microdilution checkerboard methodology. The ultrastructural alterations of S. schenckii after exposure to the combination of ibuprofen and amphotericin B were evaluated by scanning electron microscopy (SEM) and flow cytometry analysis. As a single agent, ibuprofen inhibited Sporothrix growth with a MIC median of 256 μg/mL, while the MIC medians of ibuprofen in combination with antifungals were 16 μg/mL and 128 μg/mL. The MIC values of amphotericin B, itraconazole, and terbinafine were reduced when isolates were co-incubated with ibuprofen, mainly the polyene. The major alteration after treatment with the ibuprofen/amphotericin B combination was the increase in the presence of filamentous forms and high membrane damage with loss of plasma membrane integrity. In summary, we demonstrated that ibuprofen increases the in vitro activity of antifungals, mainly amphotericin B, against S. brasiliensis and S. schenckii. Future in vivo studies exploring combination therapy with ibuprofen and antifungals in animal models are needed to confirm its efficacy.

Sporotrichosis is a neglected endemic mycosis with a high incidence in Latin America, mainly in Brazil. Sporothrix schenckii is the most frequent species in Latin America, whereas Sporothrix brasiliensis is the predominant species observed in Brazil and is associated with both human and animal sporotrichosis. Sporotrichosis treatment remains restricted to a few options, itraconazole being the first choice for human and animal therapy. In this work, we screened the molecular library Pathogen Box (Medicines for Malaria Venture [MMV], Switzerland) in search of compounds with anti-Sporothrix activity. Our initial screen of the 400 compounds identified five compounds that inhibited more than 80% of S. brasiliensis and S. schenkii growth. Among those, three compounds (MMV675968, MMV102872, and MMV002817 (known as iodoquinol)) not previously described as antifungals or agrochemicals, were selected for further evaluation. MMV102872 and iodoquinol showed the most promising combination of antifungal activity (lower inhibitory concentration) and fungal selectivity (lower cytotoxicity in LLC-MK2 cells). Scanning electron microscopy and flow cytometry analyses revealed that MMV102872 and iodoquinol induced changes in cell morphology, membrane integrity, and the presence of neutral lipids, impairing fungal survival. Our results indicate that MMV102872 and iodoquinol are promising molecules for use as scaffolds for the development of new antifungal agents., Competing Interests: The authors have declared that no competing interests exist.

The new complexes Zn(ITZ) 2 Cl 2 (1) and Zn(ITZ) 2 (OH) 2 (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, 1 H and 13 C{ 1 H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis , Trypanosoma cruzi , and Toxoplasma gondii , and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii The Zn-ITZ complexes exhibited a broad spectrum of action, with antiparasitic and antifungal activity in low concentrations. The strategy of combining zinc with ITZ was efficient to enhance ITZ activity since Zn-ITZ-complexes were more active than the azole alone. This study opens perspectives for future applications of these Zn-ITZ complexes in the treatment of parasitic diseases and sporotrichosis., (Copyright © 2020 American Society for Microbiology.)

Naphthoquinones are the most commonly occurring type of quinones in nature. They are a diverse family of secondary metabolites that occur naturally in plants, lichens and various microorganisms. This subgroup is constantly being expanded through the discovery of new natural products and by the synthesis of new compounds via innovative techniques. Interest in quinones and the search for new biological activities within the members of this class have intensified in recent years, as evidenced by the evaluation of the potential antimicrobial activities of quinones. Among fungi of medical interest, yeasts of the genus Candida are of extreme importance due to their high frequency of colonization and infection in humans. The objective of this review is to describe the development of naphthoquinones as antifungals for the treatment of Candida species and to note the most promising compounds. By using certain criteria for selection of publications, 68 reports involving both synthetic and natural naphthoquinones are discussed. The activities of a large number of substances were evaluated against Candida albicans as well as against 7 other species of the genus Candida. The results discussed in this review allowed the identification of 30 naphthoquinones with higher antifungal activities than those of the currently used drugs.

Purpose: Sporothrix brasiliensis, the most virulent species in the Sporothrix schenckii complex, is responsible for the ongoing epidemics of human and animal sporotrichosis in Brazil. Feline outbreaks are usually driven by S. brasiliensis and followed by extensive transmission to humans. Itraconazole is the first-line treatment for both feline and human sporotrichosis; however, reduced sensitivity is an emerging issue. Thus, we investigated the effect of the widely used antifungal clotrimazole - alone or in combination with itraconazole - against the pathogenic (yeast) form of feline and human S. brasiliensis isolates, in vitro., Methodology: Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values were determined for treatment with clotrimazole and itraconazole, as monotherapy or in combination. In addition, the effect of the drugs on neutral lipid levels and the yeast ultrastructure were evaluated by flow cytometry and transmission electron microscopy (TEM), respectively., Results: The MIC and MFC values show that clotrimazole was more effective than itraconazole against feline S. brasiliensis isolates, while human isolates were more sensitive to itraconazole. Similarly to itraconazole, treatment with clotrimazole induced statistically significant neutral lipid accumulation in S. brasiliensis yeasts, and treated yeasts displayed irregularities in the cell membrane and a thicker cell wall when observed by TEM. Clotrimazole increased the antifungal activity of itraconazole in combination assays, with a synergistic effect for two feline isolates., Conclusion: The strong activity of clotrimazole against feline S. brasiliensis isolates suggests that this drug is potentially a new alternative for the treatment of feline sporotrichosis, alone or in combination with itraconazole.

Clinical and Laboratory Standards Institute (CLSI) conditions for testing the susceptibilities of pathogenic Sporothrix species to antifungal agents are based on a collaborative study that evaluated five clinically relevant isolates of Sporothrix schenckii sensu lato and some antifungal agents. With the advent of molecular identification, there are two basic needs: to confirm the suitability of these testing conditions for all agents and Sporothrix species and to establish species-specific epidemiologic cutoff values (ECVs) or breakpoints (BPs) for the species. We collected available CLSI MICs/minimal effective concentrations (MECs) of amphotericin B, five triazoles, terbinafine, flucytosine, and caspofungin for 301 Sporothrix schenckii sensu stricto , 486 S. brasiliensis , 75 S. globosa , and 13 S. mexicana molecularly identified isolates. Data were obtained in 17 independent laboratories (Australia, Europe, India, South Africa, and South and North America) using conidial inoculum suspensions and 48 to 72 h of incubation at 35°C. Sufficient and suitable data (modal MICs within 2-fold concentrations) allowed the proposal of the following ECVs for S. schenckii and S. brasiliensis , respectively: amphotericin B, 4 and 4 μg/ml; itraconazole, 2 and 2 μg/ml; posaconazole, 2 and 2 μg/ml; and voriconazole, 64 and 32 μg/ml. Ketoconazole and terbinafine ECVs for S. brasiliensis were 2 and 0.12 μg/ml, respectively. Insufficient or unsuitable data precluded the calculation of ketoconazole and terbinafine (or any other antifungal agent) ECVs for S. schenckii , as well as ECVs for S. globosa and S. mexicana These ECVs could aid the clinician in identifying potentially resistant isolates (non-wild type) less likely to respond to therapy., (Copyright © 2017 American Society for Microbiology.)

Calcineurin inhibitors - such as the clinically used drug tacrolimus - are active against important fungal pathogens, particularly when combined with azoles. However, tacrolimus has not been tested against sporotrichosis, an endemic subcutaneous mycosis with worldwide distribution. Here, we evaluated the activity of tacrolimus and cyclosporine A in vitro - as monotherapy and in combination with itraconazole or fluconazole - against yeasts of Sporothrix brasiliensis and S. schenckii , the main sporotrichosis agents in Brazil. We also analyzed the effect of tacrolimus treatment on intracellular neutral lipid levels, which typically increase after azole treatment. Tacrolimus inhibited the growth of yeasts from S. brasiliensis and S. schenckii reference isolates, with minimum inhibitory concentration (MIC) values (required for ≥50% growth inhibition) of 1 and 2 mg/L, respectively. Importantly, the combination of tacrolimus and azoles exhibited high synergy toward reference Sporothrix isolates. Tacrolimus combined with itraconazole significantly increased neutral lipid accumulation in S. brasiliensis , but not in S. schenckii . Clinical isolates of S. brasiliensis and S. schenckii were more sensitive to tacrolimus as monotherapy than feline-borne isolates, however, synergy between tacrolimus and azoles was only observed for feline-borne isolates. Cyclosporine A was effective against S. brasiliensis and S. schenckii as monotherapy (MIC = 1 mg/L), but exhibited no synergy with itraconazole and fluconazole. We conclude that tacrolimus has promising antifungal activity against sporotrichosis agents, and also increases the activity of the current anti-sporotrichosis therapy (itraconazole and fluconazole) in combination assays against S. brasiliensis feline-borne isolates.

Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells.

Inhibition of Δ(24)-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker(®) Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.

Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine - already in clinical use against leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values of 1-2 µg ml(-1). Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed a decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers and accumulation of an electron-dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine was comparable to those of amphotericin B, but miltefosine showed a higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis., (© 2015 The Authors.)