Clinical and Laboratory Standards Institute (CLSI) conditions for testing the susceptibilities of pathogenic species to antifungal agents are based on a collaborative study that evaluated five clinically relevant isolates of 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 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, 486 , 75, and 13 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 and , 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 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 , as well as ECVs for and These ECVs could aid the clinician in identifying potentially resistant isolates (non-wild type) less likely to respond to therapy.

The in vitro activity of the antifungal agents amphotericin B (AMB), itraconazole (ITC), posaconazole (PSC), voriconazole (VRC), and terbinafine (TRB) against 32 Brazilian isolates of Sporothrix brasiliensis, including 16 isolates from a recent (2011-2012) epidemic in Rio de Janeiro state, was examined. We describe and genotype new isolates and clustered them with 16 older (from 2004 or earlier) S. brasiliensis isolates by phylogenetic analysis. We tested both the yeast and the mycelium form of all isolates using broth microdilution methods based on the reference protocols M38-A2 and M27-A3 (recommended by the Clinical and Laboratory Standards Institute). Considering minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), TRB was found to be the most active drug in vitro for both fungal forms, followed by PSC. Several isolates showed high MICs for AMB and/or ITC, which are currently used as first-line therapy for sporotrichosis. VRC displayed very low activity against S. brasiliensis isolates. The primary morphological modification observed on treated yeasts by transmission electron microscopy analysis was changes in cell wall. Our results indicate that TRB is the antifungal with the best in vitro activity against S. brasiliensis and support the use of TRB as a promising option for the treatment of cutaneous and/or lymphocutaneous sporotrichosis.

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.

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 mg 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 antimelanin 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. INTRODUCTIONSporotrichosis is the most frequent subcutaneous mycosis in Latin America, and has high incidence rates in Brazil, especially in the Rio de Janeiro state, where zoonotic transmission prevails (Barros et al., 2010). This disease is caused by dimorphic fungal species from the Sporothrix schenckii complex (Marimon et al., 2007;, including Sporothrix brasiliensis, the most virulent species (ArrillagaMoncrieff et al., 2009;Fernandes et al., 2013) and the most prevalent in the clinical cases from the Rio de Janeiro state (Oliveira et al., 2011;Rodrigues et al., 2013a;Borba-Santos et al., 2014). In zoonotic transmission, sporotrichosis is mainly acquired by scratches, bites or direct contact with lesion secretion from contaminated animals, where yeasts are the infective forms (Rodrigues et al., 2013b).The 'gold standard' treatment for cutaneous and lymphocutaneous forms of sporotrichosis is itraconazole, while amphotericin B is the first-line drug of choice for disseminated forms of the disease (Kauffman et al., 2007). However, recent reports demonstrated that these drugs are less potent against S. brasiliensis isolates, which have high minimum inhibitory concentration (MIC) values in vitro for these antifungals (Ottonelli Stopiglia et a...

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.

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.

Inhibition of Δ24-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.

Metal–antifungal drug complexes were investigated against fungus causing of sporotrichosis. They were more active against fungal cells than to mammalian cells.