Antifungal activity of silver salts of Keggin-type heteropolyacids against Sporothrix spp.

Sporotrichosis is a chronic and subacute mycosis causing epidemiological outbreaks involving sick cats and humans in southeastern Brazil. The systemic disease prevails in cats, and in humans, the symptoms are restricted to skin in immunocompetent individuals. Under these conditions, the prolonged treatment of animals and cases of recurrence justify the discovery of new treatments for sporotrichosis. This work addresses the antifungal activity of silver salts of Keggin-type heteropolyacid salts (Ag-HPA salts) such as Ag3[PW12O40], Ag6[SiW10V2O40], Ag4[SiW12O40] and Ag3[PMo12O40] and interactions with the antifungal drugs itraconazole (ITC), terbinafine (TBF) and amphotericin B (AMB) on the yeast and mycelia forms of Sporothrix spp. Sporothrix spp. yeast cells were susceptible to Ag-HPA salts at minimum inhibitory concentration (MIC) values ranging from 8 to 128 µg/mL. Interactions between Ag3[PW12O40] and Ag3[PMo12O40] with itraconazole and amphotericin B resulted in higher antifungal activity with a reduction in growth and melanization. Treated cells showed changes in cell membrane integrity, vacuolization, cytoplasm disorder, and membrane detachment. Promising antifungal activity for treating sporotrichosis was observed for the Ag-HPA salts Ag3[PMo12O40] and Ag3[PW12O40], which have a low cost, high yield and activity at low concentrations. However, further evaluation of in vivo tests is still required.

Potassium iodide and miltefosine inhibit biofilms of Sporothrix schenckii species complex in yeast and filamentous forms.

This study aimed to evaluate the yeast biofilm growth kinetics and ultrastructure of Sporothrix schenckii complex and assess their mature biofilm susceptibility in filamentous and yeast forms to potassium iodide (KI) and miltefosine (MIL). Yeast biofilms were evaluated by crystal violet staining, XTT reduction assay and microscopic techniques. Susceptibility of planktonic and sessile cells was analyzed by broth microdilution. S. schenckii complex in yeast form produced biofilms, with an optimum maturation at 96 h, showing multilayered blastoconidia embedded in extracellular matrix. KI and MIL minimum inhibitory concentration (MIC) ranges against planktonic cells were 62,500-250,000 µg/ml and 0.125-4 µg/ml, respectively. KI and MIL reduced biofilm metabolic activity by 75.4% and 67.7% for filamentous form and 55.1% and 51.6% for yeast form, respectively. This study demonstrated that S. schenckii complex forms biofilms in vitro, and potassium iodide and miltefosine inhibit Sporothrix spp. biofilms in both filamentous and yeast forms.

Generation of Sporothrix schenckii mutants expressing the green fluorescent protein suitable for the study of host-fungus interactions.

Sporotrichosis is an infection caused by members of the Sporothrix genus, and among them, Sporothrix schenckii is one of the etiological agents. Both, the disease and the causative agent have gained interest in the recent years, because of the report of epidemic outbreaks, and the description of the disease transmission from animals to human beings. Despite the relevance of S. schenckii in the clinical field, there are basic aspects of its biology poorly explored. So far, Agrobacterium tumefaciens-mediated transformation has been reported as an alternative for genetic manipulation of this fungal pathogen. Here, we report the optimization of the transformation method and used this to generate insertional mutants that express the green fluorescent protein in S. schenckii. We obtained five mutant strains that showed mitotic stability and expression of the reporter gene. The strains displayed normal cell wall composition, and a similar ability to interact ex vivo with human monocytes and monocyte-derived macrophages. Moreover, the virulence in larvae of Galleria mellonella was similar to that obtained with the wild-type control strains. These data indicate that these fluorescent mutants with normal ability to interact with the host could be used in bioimaging to track the host-Sporothrix interaction in vivo.

Origin and distribution of Sporothrix globosa causing sapronoses in Asia.

PURPOSE: The aim of the study was to evaluate the main sources and epidemiological patterns and speculate on the evolutionary origin of Sporothrix globosa in Asia. METHODOLOGY: Case and case series literature on sporotrichosis in Asia from January 2007 onwards were reviewed using meta-analysis. Phylogenetic analysis of relevant S. globosa was carried out on the basis of concatenated sequences of ITS, TEF3 and CAL. A haplotype network of CAL sequences of 281 Sporothrix isolates was analysed to determine the population structure of S. globosa. RESULTS: Nearly all cases of sporotrichosis caused by S. globosa in Asia were human. In contrast to the remaining pathogenic Sporothrix species, feline transmission was exceptional; nearly all regional cat-associated cases were caused by Sporothrix schenckii. While the latter species was highly variable and showed recombination, S. globosa seemed to be a clonal offshoot, as was Sporothrix brasiliensis. The origin of the segregants was located in an area of high variability in S. schenckii with a relatively high frequency of Asian strains. CONCLUSION: In Asia, S. globosa was the prevalent species. The low diversity of S. globosa suggested a recent divergence with a founder effect of low variability from the variable ancestral species, S. schenckii.

Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine.

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.

Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine

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.

Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or 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.

Susceptibility of Sporothrix brasiliensis isolates to amphotericin B, azoles, and terbinafine.

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.

Biotreatment of a gas-phase volatile mixture from fibreglass and composite manufacturing industries.

Acetone, toluene and styrene (ATS) are representative air pollutants emanating during the production process in fibreglass and composite manufacturing industries. In this study, the performance of a steady-state biofilter inoculated with the fungus Sporothrix variecibatus was tested at different empty bed residence times (EBRTs), and at different inlet concentrations of ATS, corresponding to total pollutant loading rates ranging from 30 to 490 gm(-3)hour(-1). Styrene was somewhat better removed (47-100%) in the biofilter than acetone (34-100%) and toluene (42-100%), with maximum elimination capacities (EC(max)) of 108, 72 and 144 gm(-3)hour(-1), for ATS, respectively. Besides, it was observed that, although increasing the concentration of ATS decreased their removal, the presence of toluene also decreased the EC(max) of both acetone and toluene in the ternary mixture. During transient operations, the biofilter was subjected to intermittent shutdown and re-start operations where the gas-phase pollutant flow was stopped for either 5 or 16d. It was observed that, for longer shutdown periods (16d), the biofilter required nearly 8-10d to reach similar removal patterns to those observed before the shutdown phase. Batch biodegradation tests were conducted, using Sporothrix-like microorganisms present in the leachate of the biofilter, with a mixture of ATS as the sole carbon source. Complete removal of ATS was observed within the test period of 168 hours. Styrene was degraded faster, with a specific substrate utilization rate of 0.9 mg styrenemg biomass(-1)hour(-1), followed by toluene (0.6) and acetone (0.44). The effectiveness of the biofilter to reach high total EC (321.3 gm(-3)hour(-1)), and withstand transient operations shows the robustness of this fungal-bioreactor and its suitability to handle emissions from a fibreglass and composite manufacturing industry.

Sporothrix schenckii complex and sporotrichosis, an emerging health problem.

Sporothrix schenckii, now named the S. schenckii species complex, has largely been known as the etiological agent of sporotrichosis, which is an acute or chronic subcutaneous mycosis of humans and other mammals. Gene sequencing has revealed the following species in the S. schenckii complex: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. The increasing number of reports of Sporothrix infection in immunocompromised patients, mainly the HIV-infected population, suggests sporotrichosis as an emerging global health problem concomitant with the AIDS pandemic. Molecular studies have demonstrated a high level of intraspecific variability. Components of the S. schenckii cell wall that act as adhesins and immunogenic inducers, such as a 70-kDa glycoprotein, are apparently specific to this fungus. The main glycan peptidorhamnomannan cell wall component is the only O-linked glycan structure known in S. schenckii. It contains an α-mannobiose core followed by one α-glucuronic acid unit, which may be mono- or di-rhamnosylated. The oligomeric structure of glucosamine-6-P synthase has led to a significant advance in the development of antifungals targeted to the enzyme's catalytic domain in S. schenckii.

Sporotrichosis caused by Sporothrix globosa in Rio De Janeiro, brazil: case report.

This report describes the first isolation of Sporothrix globosa from a Brazilian patient. A 77-year-old woman was examined for sporotrichosis infection. Histopathological examination of skin biopsy revealed chronic granulomatous infiltrate with microabcess. Furthermore, S. schenckii-like yeasts were evident as demonstrated by PAS and Grocott stains. The fungus was identified based on colony morphology on Sabouraud Dextrose Agar slants, Potato Dextrose Agar, and Corn Meal Agar, microscopic morphology on slides cultures, and assimilation of different carbon sources. The species confirmation was made by molecular methodology.

Biodegradation of gas-phase styrene using the fungus Sporothrix variecibatus: impact of pollutant load and transient operation.

Biofiltration of gas-phase styrene was studied using a newly isolated fungus Sporothrix variecibatus, in a perlite biofilter, at inlet concentrations and gas-flow rates ranging from 0.13 to 14 g m(-3) and 0.075 to 0.34 m(3) h(-1), respectively, corresponding to empty bed residence times (EBRT) ranging between 91 and 20s. Styrene loading rates were varied between 50 and 845 g m(-3) h(-1)and a maximum elimination capacity of 336 g m(-3) h(-1) was attained with nearly 65% styrene removal. On the other hand, the critical inlet loads to achieve more than 90% removal were 301, 240 and 92 g m(-3) h(-1) for EBRT of 91, 40, and 20s, respectively. In order to test the stability and shock bearing capacity of the fungal biofilter, short-term tests were conducted by suddenly increasing the gas-phase styrene concentration, while maintaining the gas-flow rate constant. The response, a restoration in the removal performance to previous high values, after subjecting the biofilter to shock loads proves the resilient nature of the attached Sporothrix sp. and its suitability for biofiltration under non-steady state conditions.

[Sporotrichosis and dematiaceous fungal skin infections].

Sporotrichosis is a chronic infectious granuloma of skin. The detection of fungal elements in pathological examination and the isolation of Sporothrix schenckii from the lesion are requisite for diagnosis. The sporotrichin test is useful as an auxiliary examination, but a false-negative reaction might occur in some cases. Oral potassium iodide is first choice of treatment, because of its modest cost and usefulness, although gastrointestinal disorder is a frequent side effect. Itraconazole should be the second selection, and then terbinafine. Local thermotherapy is also effective as an additional therapy. Dematiaceous fungal skin infections are divided into two groups by their parasitic form, chromoblastomycosis and phaeohyphomycosis. Chromoblastomycosis is also called chromomycosis in Japan. It is most important for clinical diagnosis to detect dark brown spores in the scale of chromoblastomycosis and dark brown hyphae in the pus of phaeohyphomycosis by microscopic examination. Both morphological and molecular biological approaches are recommended for identification of fungi. In treatment, the drug appropriate in each case should be selected, and the combination of surgical excision, local thermotherapy, laser therapy or cryotherapy must be considered.

[Sporotrichosis as the cause of a leg ulcer]. / Sporotrichose als Ursache eines Ulcus cruris.

A young man presented at Lugala Lutheran Hospital (Tanzania) with an ulcer on his lower leg which had developed over the past 9 weeks. Subcutaneous nodules and plaques were found all the way up to his groin; this observation prompted a strong suspicion that the patient had the lymphocutaneous form of sporotrichosis which had not been seen at this hospital before. The diagnosis was confirmed histopathologically. The patient was then treated with saturated solution of potassium iodide.

Conjunctival sporotrichosis in the absence of antecedent trauma.

PURPOSE: To describe a patient with conjunctivitis. METHODS: Case report and review of the literature. RESULTS: A 34-year-old cattle farmer with no history of trauma developed gradual onset of an inflamed bulbar conjunctival mass associated with ipsilateral preauricular lymphadenopathy. Excisional biopsy of the conjunctival mass and subsequent histopathologic examination revealed suppurative granulomatous inflammation and small budding yeasts. Electron microscopy confirmed the presence of small intracellular and extracellular yeast organisms. Cultures grew the dimorphic fungi Treatment with oral itraconazole and topical fluconazole resulted in complete resolution of the infection. CONCLUSION: characteristically infects the skin and regional lymphatics after penetrating trauma, but atraumatic infection of pulmonary mucosal surfaces can occur upon inhalation of the fungal spores. Our case report suggests that atraumatic exposure to may be sufficient to establish conjunctival infection.

Are giant cells conidia in Sporothrix schenckii? -Freeze-fracture electron microscopic observation-.

Sporothrix schenckii is a well-known pathogenic dimorphic fungus. In this study, we focused on the plasma membrane ultrastructures of giant cells of S. schenckii seen mainly on Sabouraud's dextroseagar slant medium. In the organisms grown for 1, 2, 3, 4, 6 and 8 weeks at 27 and 37 degrees C on brain heart infusion and Sabouraud's dextrose agar slant media, the number of conidia, hyphae, brownish and non-brownish giant cells were counted in ten separated areas under light microscope (x100) to determine the culture conditions under which giant cells were generated. The results showed that brownish giant cells were predominantly seen after longer cultivation periods. Using freeze-fracture electron microscopy, larger oval- or round shaped cells can be identified as conidia by their plasma membrane ultrastructure characteristics, i.e, trench-like invaginations seen in ordinary mature conidia (Maeda M et al.; Can J Microbiol 33: 40, 1987). From these structural features seen by freeze-fracture electron microscopy, giant cells appeared possibly be conidia and were suggested to be starved because of their predominant existence under longer cultivation conditions.

Disseminated sporotrichosis with extensive cutaneous involvement in a patient with AIDS.

Sporotrichosis most commonly presents as a localized, lymphocutaneous infection that follows trauma, such as an injury from a rose thorn. In patients infected with HIV, it may be widespread and disseminated. We describe a patient with AIDS who developed disseminated sporotrichosis, a rare opportunistic fungal infection that may affect these patients. The condition remained undiagnosed because of failure to recognize characteristic histopathologic findings and failure of clinicians to interface closely with the microbiology laboratory. The condition was difficult to treat, requiring systemic administration of amphotericin. While localized sporotrichosis is an innocuous disorder that responds well to therapy, in immunocompromised hosts, it is potentially life-threatening and may require prolonged therapy with potentially toxic medications such as amphotericin B. It is important that clinicians be aware of the presentation of this unusual opportunistic infection and that they maintain close communication with pathology and clinical microbiology laboratories to ensure that proper stains and cultures are performed to avoid potential misdiagnosis.

Differences in virulence of Sporothrix schenckii conidia related to culture conditions and cell-wall components.

A murine model was used to evaluate the virulence of Sporothrix schenckii conidia cultured for 4, 7, 10 or 12 days in Sabouraud's dextrose broth (SDB). A correlation was observed between length of culture and virulence. Mice infected intravenously with S. schenckii conidia cultured for 4 or 7 days showed 40-100% cumulative mortality. In contrast, mice infected with conidia from cultures grown for 10 or 12 days in SDB showed no mortality (100% survival). A much greater accumulation of fungal colony forming units (cfu) was observed in the lungs, livers and spleens of mice inoculated with conidia of S. schenckii cultured for 7 days than in mice infected with conidia cultured for 12 days. The livers of mice from the former group showed a widespread granulomatous reaction whereas mice inoculated with S. schenckii cultured for 12 days showed a more limited response with fewer granulomas. No difference in viability or replicative capacity was discerned for these S. schenckii cultured cells. However, the more virulent forms of the fungus showed differences in cell-wall sugar composition with rhamnose:mannose molar ratios of 1.7:1.0 for cells cultured for 4 days and 1.0:1.7 for conidia cultured for 12 days. These results suggest that the virulence of S. schenckii conidia may be determined by their cell-wall composition.

Elastin fibers resembling Sporothrix schenkii in the skin of a patient with acquired immunodeficiency syndrome.

We describe a patient with the acquired immunodeficiency syndrome who presented with an erythematous skin rash on his trunk and extremities. Initial histologic examination of a skin biopsy revealed silver-staining elements resembling Sporothrix schenkii. Additional histochemical and ultrastructural studies revealed that these elements were elastin fibers rather than fungi. The literature describing pseudofungal infections is reviewed.

X-ray scanning microanalysis of Sporothrix schenckii.

X-ray scanning microanalysis was applied to the study of fungal samples such as Sporothrix schenckii. This technique is sufficiently sensitive to detect many elements in the periodical classification of Mendeleev.