Hyperthermia is a promising potential adjunct to treating sporotrichosis: A refractory case of HIV sporotrichosis and in vitro experiments on Sporothrix spp.

BACKGROUND: Hyperthermia is a common monotherapy for sporotrichosis, but only in patients with special conditions, such as pregnancy and nursing. However, hyperthermia has not been used more widely for sporotrichosis in clinical practice. PATIENTS/METHODS: An HIV-positive adult male with lymphocutaneous sporotrichosis caused by Sporothrix globosa that did not respond to conventional itraconazole therapy lasting >2 months received adjunctive therapy with local hyperthermia. To simulate the effects of heat exposure on the growth and morphology of Sporothrix spp. in vitro, S. globosa, S. schenckii and S. brasiliensis were exposed to intermittent heat (42°C) for 1 h a day for 7 or 28 days and observed under transmission electron microscopy. RESULTS: Itraconazole combined with local hyperthermia significantly improved the lesions, and the patient was successfully cured of sporotrichosis, with no recurrence after 2 years of follow-up. Cultures of Sporothrix spp. treated with 7 days of daily heat exposure in vitro showed obvious decreases in colony diameters, but not numbers, compared with untreated cultures (p < .001). After 28 days of heat exposure in vitro, Sporothrix spp. were unable to thrive (p < .001), and ultrastructural alterations, including loose cell wall structure, incomplete cell membrane, disrupted vacuoles and fragmented nuclei, were noticeable. CONCLUSIONS: Our case findings and in vitro experiments on Sporothrix spp., together with a literature review of previous sporotrichosis cases, suggest that hyperthermia has a clinical role as a treatment adjunct. Large-scale clinical trials are required to examine the utility of hyperthermia in various forms of cutaneous sporotrichosis.

Nikkomycin Z for the treatment of experimental sporotrichosis caused by Sporothrix brasiliensis.

Sporotrichosis caused by Sporothrix brasiliensis is a global emergent infectious disease. Due to the scarcity of therapeutic options for fungal diseases, new antifungals are urgently needed. Nikkomycin Z (NikZ) is a future option as an agent against dimorphic fungi. We evaluated NikZ monotherapy and in combination with itraconazole (ITZ; the conventional therapy) in the treatment of experimental sporotrichosis caused by S. brasiliensis in a murine model. Animals were subcutaneously infected, and treated orally for 30 days. The study groups were as follows control (untreated), ITZ group (50 mg/kg/day), and three groups treated with NikZ, two by monotherapy (200 or 400 mg/kg/day), and one combining NikZ (400 mg/kg/day) and ITZ. Efficacy of treatments was evaluated via body weight gain, mortality and fungal burden in tissues. Efficacy was noted in all treatment groups, and the group receiving the drug combination showed even better results than those with monotherapy. Our study shows for the first time the high potential of NikZ to be used in the treatment of sporotrichosis caused by S. brasiliensis.

Sporotrichosis in dogs: epidemiological and clinical-therapeutic profile and the emergence of itraconazole-resistant isolates.

Sporotrichosis is a neglected and emerging mycosis caused by the traumatic implantation of Sporothrix propagules into the (sub)cutaneous tissues of humans and animals. We evaluated canine sporotrichosis's clinical-therapeutic, epidemiological profile, and in vitro susceptibility of isolates to itraconazole. The variables were evaluated by a chi-square test. A total of 69 dogs were infected with Sporothrix spp., and the molecular identification revealed an overwhelming occurrence of S. brasiliensis as the etiological agent. The epidemiological profile was male (56.5%), adults (4.9 ± 1.92 years old; 69.6%), and mongrels (53.6%). The clinical signs were 76.8%, ulcers, draining tracts, and nodules were predominant, mainly in the nasal region (82.2%). Dogs were diagnosed late with an evolution time of up to 3 months (34.8%). According to the prior therapeutic information, 52.2% received empirical therapy, 79.2% antibiotics, and had a 0.29 significantly greater chance of presenting lesion evolution time ˃ 3 months (P < .05; Odds Ratio [OR] 1/0.29). Additionally, 25 S. brasiliensis isolates recovered between 2006-2012 (n = 15; Minimal inhibitory concentration (MIC): 0.06-2 µg/ml) and 2013-2018 (n = 10; MIC: 2→16 µg/ml) were tested against itraconazole (ITZ). These findings highlighted the resistance to ITZ in clinical cases due to S. brasiliensis occurring after 2013, showing the temporal evolution of ITZ-resistance. We warn of the importance of accurate and early diagnosis in Sporothrix-affected areas, and we report the emergence of ITZ-resistant isolates in Southern Brazil.

Sporotrichosis is a fungal zoonosis. We investigated the clinical-therapeutic, epidemiological profile, and in vitro susceptibility of isolates to itraconazole (ITZ) in canine cases in Southern Brazil. Our study highlighted the emergence of ITZ-resistant Sporothrix brasiliensis and the main challenges for clinical control of this neglected disease.

Feline sporotrichosis: a case series of itraconazole-resistant Sporothrix brasiliensis infection.

The treatment of feline sporotrichosis is a challenge for veterinary clinicians since refractory cases may occur, due either to patient and/or to pharmacological management errors or due to the development of antifungal resistance. Thus, we aimed to describe the therapeutic history of feline cases infected by itraconazole-resistant Sporothrix brasiliensis in an endemic region of Southern Brazil. Medical records of cats attended at the Veterinary Clinic Hospital (Pelotas/RS, Brazil) between 2016 and 2017 were reviewed. Twelve cases of infection by S. brasiliensis with that showed high minimum inhibitory concentration (MIC) values (≥ 4 µg/mL) to itraconazole by M38-A2 of CLSI were selected. At the hospital consultation, disseminated (cats 1-l0, 12) and localized (cat 11) skin lesions remained in the cats, even after treatment with fluconazole, ketoconazole (02/12), and itraconazole (ITZ, 09/12) performed before this study. High doses (25-100 mg/kg/day) of ITZ for up to 4 months (03/12, cats 2, 6, 12) or over 12 months (05/12, cats 1, 5, 7, 8, 11) did not provide a clinical cure, except for the association of ITZ plus potassium iodide (01/12, cat 12) for 3 months, which proved useful in infections with itraconazole-resistant S. brasiliensis. However, the combined issues of abandonment of therapy by owners for financial reasons, difficulties surrounding therapy administration (03/12, cats 6, 11, 12), and the inappropriate choice of medication (01/12, cat 6), together reflect the reality of this endemic region, which greatly compromises clinical healing. This study highlighted the occurrence of refractory cases by itraconazole-resistant S. brasiliensis in cats from Southern Brazil, as well as the abandonment of treatment and therapeutic errors. We warn of the need for antifungal susceptibility tests to adapt therapeutic protocols in feline sporotrichosis.

The clinico-epidemiological characteristics and therapeutic experience of 152 patients with cutaneous sporotrichosis: a 10-year retrospective study from India.

BACKGROUND: Cutaneous sporotrichosis, a subcutaneous mycosis because of Sporothrix schenckii, is sporadic worldwide with local hyperendemic pockets. OBJECTIVES: To study clinico-epidemiological and therapeutic aspects of sporotrichosis in our clinic. METHODS: We retrospectively analyzed medical records of 152 (M:F 52:100) patients with cutaneous sporotrichosis managed during 2010-2019. RESULTS: All patients were involved in agricultural activities, and 63.2% were aged 21-60 years. Women outnumbered men by nearly two times. Fixed and lymphocutaneous sporotrichosis occurred in 54.6% and 43.4% patients, respectively. Only 2% of patients had multifocal sporotrichosis. Only 48% of patients imputed their disease to prior injuries. Extremities, upper in 53.9% and lower in 21% of patients, were mostly involved. Scrotum involvement in one patient was unusual. A mixed inflammatory infiltrate in 38.7%, chronic granuloma formation in 35%, and presence of spores in 48.9% biopsies was noted. S. schenckii grew on Sabouraud's dextrose agar in 40.2% of cases. Treatment with saturated solution of potassium iodide was curative in 76.8% patients, and lesions healed in 2-9 months (average 5.2 months). Metallic taste was experienced by 42.9% of patients. Itraconazole therapy was safe and effective in seven patients, and the response was better when combined with SSKI compared to either drug used alone. CONCLUSION: Cutaneous sporotrichosis mostly affects persons during active years of life. The injuries predisposing to infection are mostly forgotten. Both fixed and lymphocutaneous sporotrichosis involving extremities remain common forms. SSKI alone or in combination with itraconazole is safe and effective treatment. Itraconazole is preferable in patients having preexisting hypothyroidism or intolerance to SSKI.

5-Aminolevulinic acid-mediated photodynamic therapy has effective antifungal activity against Sporothrix globosa in vitro.

BACKGROUND: An alternative therapy for sporotrichosis is necessary to reduce the treatment time and raise clinical efficacy. The 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is a promising platform with which to treat mycoses. However, despite the worldwide prevalence of Sporothrix globosa, a causative agent of sporotrichosis, the effect of ALA-PDT on this pathogen has not been validated. OBJECTIVES: The aim of this study was to evaluate the effect of ALA-PDT on S globosa and the protection of melanin through an in vitro study. The mechanisms involved were also investigated. METHODS: To estimate the survival rate of S globosa treated with ALA-PDT and the protection offered by melanin, the conidia and yeast cells of wild-type S globosa (Mel+), other clinical strains, tricyclazole-treated Mel+ and an albino mutant strain (Mel-) were incubated with and without ALA or irradiation. Reactive oxygen species generation by Mel+ conidia induced by ALA-PDT was assayed. SEM and TEM were conducted to obverse ultrastructural changes in the conidia. A comet assay was performed to evaluate DNA damage. RESULTS: The survival rate of S globosa conidia and yeast cells significantly decreased following incubation with 1.19M ALA and 162 J/cm2 irradiation in vitro. Melanin was not only capable of protecting the conidia against ALA-PDT, but also against ALA or irradiation alone. After induction by ALA-PDT, alterations in reactive oxygen species generation, DNA damage and ultrastructural changes were observed. CONCLUSIONS: ALA-PDT inhibits the survival of S globosa conidia in vitro and therefore has potential for the treatment of sporotrichosis.

Formulation and Evaluation of a Novel Itraconazole-Clotrimazole Topical Emulgel for the Treatment of Sporotrichosis.

In recent years, the development of new pharmaceutical formulations for the treatment of sporotrichosis has become a relevant research field. In this work, we aimed to develop an emulgel containing itraconazole and clotrimazole to ensure therapeutic effectiveness against Sporothrix brasiliensis. The topical use of a formulation that combines both drugs represents an interesting option for the complementary treatment of sporotrichosis. The emulgel formulation was prepared and evaluated for its zeta potential, viscosity, in vitro antifungal activity and stability at different storage conditions. The results showed that the newly developed emulgel displayed promising physicochemical characteristics, as well as a good in vitro inhibitory activity against S. brasiliensis yeasts. The results obtained in this work suggest that the emulgel containing itraconazole and clotrimazole might highly be efficient and a complementary therapy to oral administration in the treatment of sporotrichosis.

Sporotrichosis caused by Sporothrix brasiliensis in Argentina: Case report, molecular identification and in vitro susceptibility pattern to antifungal drugs.

Sporotrichosis is considered a neglected disease of humans and animals in many regions of the world and is the most frequent implantation mycosis in Latin America. OBJECTIVES: To illustrate the zoonotic importance of the disease, describing a case involving a veterinarian and an infant that acquired the disease from a domestic cat and to describe, genotype and characterize these new isolates. METHODS: Direct examination of tissue samples from the two patients and feline lesions revealed the presence of Sporothrix yeast-like organisms. Fungal cultures and molecular identification of the strains were performed. Since antifungal susceptibility data of animal-borne isolates are scarce, the in vitro susceptibility testing by a microdilution reference method was determined against azoles, amphotericin B and terbinafine. RESULTS: Fungal culture and sequence analysis of the ITS region of rDNA and calmodulin and ß-tubulin genes confirmed the diagnosis and the causative agent as Sporothrix brasiliensis. In all cases, terbinafine was the most active drug, followed by posaconazole, itraconazole and voriconazole; the least active drugs were amphotericine B and fluconazole. Lack of clinical response in the veterinarian and in the infant to itraconazole and potassium iodide, respectively was observed. CONCLUSIONS: This study contributed to the molecular epidemiology of Sporothrix species in Argentina and the characterization of the in vitro susceptibility pattern of S. brasiliensis isolates recovered from a cat and two humans involved in this case of zoonotic sporotrichosis. Bearing in mind the "One Health" concept, the experience described in the present study highlights the need for future strategies for sporotrichosis treatment, control and prevention.

Investigation of a Microemulsion Containing Clotrimazole and Itraconazole for Transdermal Delivery for the Treatment of Sporotrichosis.

The aim of this study was to develop a microemulsion (ME) formulation containing an association of itraconazole (ITC) and clotrimazole (CLT) as a transdermal delivery system for the treatment of sporotrichosis. Pseudoternary phase diagrams were constructed to optimize the ME formulation. The ME formulation selected contained 1% (w/w) ITC and 1% (w/w) CLT and was composed of 23.07% Tween® 60 (surfactant), 23.07% propylene glycol (cosurfactant/cosolvent), 30.77% benzyl alcohol (oil), and 21.09% water. The ITC/CLT-loaded ME (ITC/CLT-ME) had a droplet size value of 217 ± 0.9 nm, with a polydispersity index of 0.5 ± 0.1. Permeation experiments on pig ear skin were conducted for ITC/CLT-ME, and the results indicated that the drug permeation performance was influenced by CLT, indicating that CLT acts as a promoter enhancer. In the in vitro antifungal activity assay using Sporothrix brasiliensis yeast, the inhibition halo produced by ITC/CLT-ME exhibited a mean diameter of 43.67 ± 2.31 mm. The ITC/CLT-ME formulation did not cause skin irritation in mice. The results suggest that ITC/CLT-ME is a promising tool for the transdermal treatment of sporotrichosis.

Development of a Method for the Quantification of Clotrimazole and Itraconazole and Study of Their Stability in a New Microemulsion for the Treatment of Sporotrichosis.

Sporotrichosis occurs worldwide and is caused by the fungus Sporothrix brasiliensis. This agent has a high zoonotic potential and is transmitted mainly by bites and scratches from infected felines. A new association between the drugs clotrimazole and itraconazole is shown to be effective against S. brasiliensis yeasts. This association was formulated as a microemulsion containing benzyl alcohol as oil, Tween® 60 and propylene glycol as surfactant and cosurfactant, respectively, and water. Initially, the compatibility between clotrimazole and itraconazole was studied using differential scanning calorimetry (DSC), thermogravimetric analysis (TG), Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction (PXRD). Additionally, a simple and efficient analytical HPLC method was developed to simultaneously determine the concentration of clotrimazole and itraconazole in the novel microemulsion. The developed method proved to be efficient, robust, and reproducible for both components of the microemulsion. We also performed an accelerated stability study of this formulation, and the developed analytical method was applied to monitor the content of active ingredients. Interestingly, these investigations led to the detection of a known clotrimazole degradation product whose structure was confirmed using NMR and HRMS, as well as a possible interaction between itraconazole and benzyl alcohol.

Polar Origanum vulgare (Lamiaceae) extracts with antifungal potential against Sporothrix brasiliensis.

Oregano (Origanum vulgare) has anti-Sporothrix spp. activity, including against strains that are resistant to antifungal drugs. As the studies are limited to the essential oil, the aim of this study was to evaluate the chemical, antioxidant and cytotoxic properties of polar oregano extracts and their anti-Sporothrix brasiliensis activity. Aerial plant parts were used in the preparation of 10 min (INF10) and 60 min (INF60) infusions, a decoction (DEC) and a hydroalcoholic extract (HAE). Six phenolic acids and four flavonoids were identified and quantified through liquid-chromatography (LC-MS). Extracts in increasing order of total phenolic and flavonoid contents were HAE 40 mg/ml for the other extracts. This is the first report of oregano extracts showing antifungal activity against S. brasiliensis. Its use in the treatment of sporotrichosis may be considered upon toxicity and safe-use conditions are tested.

Efficacy of a poly-aggregated formulation of amphotericin B in treating systemic sporotrichosis caused by Sporothrix brasiliensis.

In severe cases of sporotrichosis, it is recommended to use amphotericin B deoxycholate (D-AMB) or its lipid formulations and/or in association with itraconazole (ITC). Our aim was to evaluate the antifungal efficacy of a poly-aggregated amphotericin B (P-AMB), a nonlipid formulation, compared with D-AMB on systemic sporotrichosis caused by Sporothrix brasiliensis. In vitro assays showed that Sporothrix schenckii sensu stricto and S. brasiliensis yeast clinical isolates were susceptible to low concentrations of P-AMB and D-AMB. Although P-AMB presented a higher minimal inhibitory concentration (MIC) compared to D-AMB, its cytotoxic effect on renal cells and erythrocytes was lower. For the in vivo assays, male BALB/c mice were intravenously infected with S. brasiliensis yeasts, and P-AMB or D-AMB was administered 3 days post-infection. The efficacy of five therapeutic regimens was tested: intravenous monotherapy with P-AMB or D-AMB, intravenous pulsed-therapy with P-AMB or D-AMB, and intravenous therapy with P-AMB, followed by oral ITC. These treatments increased murine survival and controlled the fungal burden in the liver, spleen, lungs, and kidneys. However, only D-AMB monotherapy or the pulsed-therapies with D-AMB or P-AMB led to 100% survival of the mice 45 days post-infection; only pulsed administration of D-AMB was able to control the fungal load in all organs 45 days post-infection. Accordingly, the histopathological findings showed reductions in the fungal burden and inflammatory reactions in these treatment regimens. Together, our results suggest that the P-AMB formulation could be considered as an alternative drug to D-AMB for treating disseminated sporotrichosis.

Fungal infection control by garlic extracts (Allium sativum L) and modulation of peritoneal macrophages activity in murine model of sporotrichosis / Controle da infecção fúngica pelos extratos de alho (Allium sativum L) e modulação da atividade de macrófagos peritoneais em modelo murino de esporotricose

Abstract Garlic (Allium sativum L.) is grown all over the world as seasoning and medicinal vegetable since 3,000 BC. Allicin is the main component of garlic, being attributed to it the most of its biological activities, such as bactericidal, antifungal and antiviral actions. However, other compounds of garlic present antioxidant, hypocholesterolemic, vasodilator activities, protective action against different types of cancer, and immunomodulatory. Fungal infections are important causes of morbidity and mortality in people mainly in immunosuppressed ones. Sporothrix schenckii, the causing agent of Sporotrichosis (most common subcutaneous mycosis in Latin America), is dimorphic fungus, of saprophytic life in soil or plants, infecting people and animals mainly through skin injuries and bruises. The main of this work was to evaluate the influence of garlic consuming on immune modulation of healthy and infected Swiss mice in induced way by S. schenckii, since these animals functioning of peritoneal macrophages as well as the nitric oxide and cytokines' production (IL-1β, IL-10 and IL-12) and to evaluate the antifungal potential of garlic with S. schenckii through minimum inhibitory concentration test and colony-forming units. The results showed that garlic offers antifungal potential with S. schenckii. The oral taking of garlic extracts influences the releasing of cytokines by macrophages, regular consuming shows anti-inflammatory effect, and its acute use may take to an inflammatory response. Mice that consumed garlic responded more effectively to fight against the infection.

Resumo O alho (Allium sativum L.) é cultivado em todo o mundo como hortaliça condimentar e medicinal desde 3.000 a. C. A alicina é o principal componente do alho, sendo atribuída a ela a maior parte das suas atividades biológicas, dentre elas as ações bactericida, antifúngica e antiviral. Porém, outros compostos do alho apresentam atividade antioxidante, hipocolesterolemiante, vasodilatadora, ação protetora contra diversos tipos de câncer e imunomoduladora. As infecções por fungos são causas importantes de morbidade e mortalidade no homem principalmente em indivíduos imunossuprimidos. O Sporothrix schenckii, agente causal da esporotricose (micose subcutânea mais comum na América Latina), é fungo dimórfico, de vida saprofítica no solo ou em vegetais, infectando homens e os animais principalmente através de lesões e arranhões na pele. O objetivo desse trabalho foi avaliar a influência do consumo de alho na imunomodulação de camundongos Swiss saudáveis e infectados de forma induzida por S. schenckii, a partir do estado funcional dos macrófagos peritoneais desses animais quanto à produção de óxido nítrico e das citocinas (IL-1β, IL-10 e IL-12) e avaliar o potencial antifúngico do alho frente ao S. schenckii por meio de teste de concentração inibitória mínima e unidades formadoras de colônia. Os resultados demonstraram que o alho apresenta potencial antifúngico frente S. schenckii. A administração oral de extratos de alho influencia a liberação de citocinas por macrófagos, o consumo regular apresenta efeito anti-inflamatório, e seu uso agudo pode gerar uma resposta inflamatória. Camundongos que consumiram alho responderam de forma mais efetiva no combate da infecção.

Fungal infection control by garlic extracts (Allium sativum L.) and modulation of peritoneal macrophages activity in murine model of sporotrichosis.

Garlic (Allium sativum L.) is grown all over the world as seasoning and medicinal vegetable since 3,000 BC. Allicin is the main component of garlic, being attributed to it the most of its biological activities, such as bactericidal, antifungal and antiviral actions. However, other compounds of garlic present antioxidant, hypocholesterolemic, vasodilator activities, protective action against different types of cancer, and immunomodulatory. Fungal infections are important causes of morbidity and mortality in people mainly in immunosuppressed ones. Sporothrix schenckii, the causing agent of Sporotrichosis (most common subcutaneous mycosis in Latin America), is dimorphic fungus, of saprophytic life in soil or plants, infecting people and animals mainly through skin injuries and bruises. The main of this work was to evaluate the influence of garlic consuming on immune modulation of healthy and infected Swiss mice in induced way by S. schenckii, since these animals functioning of peritoneal macrophages as well as the nitric oxide and cytokines' production (IL-1ß, IL-10 and IL-12) and to evaluate the antifungal potential of garlic with S. schenckii through minimum inhibitory concentration test and colony-forming units. The results showed that garlic offers antifungal potential with S. schenckii. The oral taking of garlic extracts influences the releasing of cytokines by macrophages, regular consuming shows anti-inflammatory effect, and its acute use may take to an inflammatory response. Mice that consumed garlic responded more effectively to fight against the infection.

Comparison of two in vitro antifungal sensitivity tests and monitoring during therapy of Sporothrix schenckii sensu stricto in Malaysian cats.

BACKGROUND: Feline sporotrichosis is common in Malaysia. Thermosensitivity and effects of azole treatment on fungal susceptibility are unknown. OBJECTIVES: To evaluate thermotolerance and antifungal susceptibility of feline Malaysian Sporothrix isolates, compare microdilution (MD) and E-test results, and investigate changes in susceptibility during azole therapy. METHODS: Sporothrix schenckii sensu stricto was isolated from 44 cats. Thermotolerance was determined via culture at 37°C for 7 days. Susceptibility to itraconazole (ITZ), ketoconazole (KTZ) and terbinafine (TRB) was assessed in 40 isolates by MD; to amphotericin B (AMB), KTZ, ITZ, fluconazole (FLC) and posaconazole (POS) by E-test. Results were statistically compared by Pearson's Product Moment. In eight ketoconazole treated cats, susceptibility testing to itraconazole and ketoconazole was repeated every two months for six months. RESULTS: Thermotolerance was observed in 36 of 44 (82%) isolates. Assuming that isolates growing at antifungal concentrations ≥4 mg/mL were resistant, all were resistant on E-test to FLC and AMB, 11 (28%) to POS, 6 (15%) to ITZ and 1 (3%) to KTZ. On MD, 27 of 40 (68%) were resistant to TRB, 2 (5%) to ITZ and 3 (8%) to KTZ. There was no correlation between E-test and MD results (KTZ r = 0.10, P = 0.54, and ITZ r = 0.11, P = 0.48). MD values for ITZ and KTZ did not exceed 4 mg/L during KTZ therapy. CONCLUSION: The majority of feline isolates in Malaysia are thermosensitive. Lack of correlation between E-test and MD suggests that the E-test is unreliable to test antifungal susceptibility for Sporothrix spp. compared to MD. KTZ was the antifungal drug with the lowest MIC. Prolonged KTZ administration may not induce changes in antifungal susceptibility.

Refractory sporotrichosis due to Sporothrix brasiliensis in humans appears to be unrelated to in vivo resistance.

Sporotrichosis is a subacute to chronic infection caused by members of the Sporothrix schenckii complex. Itraconazole is the first choice antifungal drug for treating this infection, with terbinafine and potassium iodide as alternatives and amphotericin B used in cases of severe infections. Correlation of antifungal susceptibility data with the clinical outcome of the patients is scarce. The aim of this study was to correlate clinical and mycological data in patients with refractory sporotrichosis. In this work, antifungal susceptibilities, determined according to the reference M38-A2 CLSI protocol, of 25 Sporothrix strains, isolated from seven human cases of sporotrichosis with adversities in the treatment, are presented. Tested drugs included itraconazole, ketoconazole, posaconazole, voriconazole, terbinafine, and amphotericin B. Fungi were identified using the T3B PCR fingerprinting. This method identified all strains as Sporothrix brasiliensis and also demonstrated a high degree of similarity between the strains. In general, voriconazole was ineffective against all strains, and elevated minimal inhibitory concentrations (MICs) were observed for amphotericin B. High itraconazole and terbinafine MICs were not observed in S. brasiliensis isolates from patients of this study. Moreover, a significant increase in itraconazole and terbinafine MIC values from strains isolated from the same patient in different periods was not observed. The results suggest that the antifungal susceptibility to terbinafine and itraconazole determined by the reference method does not play an important role in therapeutic failure of sporotrichosis and that acquisition of resistance during prolonged antifungal treatment is not likely to occur in S. brasiliensis.

Antifungal curcumin promotes chitin accumulation associated with decreased virulence of Sporothrix schenckii.

Curcumin, a yellow polyphenol compound, is known to possess antifungal activity for a range of pathogenic fungi. However, the fungicidal mechanism of curcumin (CUR) has not been identified. We have occasionally found that chitin redistributes to the cell wall outer layer of Sporothrix schenckii (S. schenckii) upon sublethal CUR treatment. Whether CUR can affect chitin synthesis via the protein kinase C (PKC) signaling pathway has not been investigated. This study describes a direct fungicidal activity of CUR against S. schenckii demonstrated by the results of a checkerboard microdilution assay and, for the first time, a synergistic effect of CUR with terbinafine (TRB). Furthermore, the results of real-time PCR showed that sublethal CUR upregulated the transcription of PKC, chitin synthase1 (CHS1), and chitin synthase3 (CHS3) in S. schenckii. The fluorescence staining results using wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC) and calcofluor white (CFW) consistently showed that chitin exposure and total chitin content were increased on the conidial cell wall of S. schenckii by sublethal CUR treatment. A histopathological analysis of mice infected with CUR-treated conidia showed dampened inflammation in the local lesion and a reduced fungal burden. The ELISA results showed proinflammatory cytokine secretion at an early stage from macrophages stimulated by the CUR-treated conidia. The present data led to the conclusion that CUR is a potential antifungal agent and that its fungicidal mechanism may involve chitin accumulation on the cell wall of S. schenckii, which is associated with decreased virulence in infected mice.

Two cases of sporotrichosis of the right upper extremity in right-handed patients with diabetes mellitus / Dos casos de esporotricosis de la extremidad superior derecha en pacientes diestros con diabetes mellitus

Background. Sporothrix species have proved to show high degrees of endemicity. Sporothrix globosa is the only pathogenic Sporothrix species that has till date been reported from China, where it is endemic in the northeastern provinces. Aims. We report two cases of lymphocutaneous sporotrichosis with diabetes mellitus as underlying disease in patients from the non-endemic area of China. Methods. A 59-year-old farmer and a 60-year-old gardener were admitted in February and June 2014, respectively. Both patients were right-handed men and presented with progressive plaques and nodules, which they had for several years, involving the right upper extremity. Skin biopsy from the granuloma was taken and cultured on Sabouraud medium, and molecular identification based on the calmodulin region was performed. Antifungal susceptibility testing was also performed with the microdilution method. Results. Biopsy of the lesions showed the presence of infectious granuloma. The fungal cultures were identified as Sporothrix globosa by conventional methods, and confirmed by molecular identification. A subsequent course of oral antifungal therapy with low dosage of itraconazole was well tolerated and resolved the infection. Conclusions. Identification of fungal species and antifungal susceptibility testing are mandatory for epidemiological and therapeutic reasons. Early diagnosis of sporotrichosis is essential to prevent those sequelae when the disease progresses and provides highly effective methods for treating this emerging disease. Avoiding the exposure to plant material potentially contaminated with fungal spores should be recommended, especially in immunocompromised patients (AU)

Antecedentes. Las especies de Sporothrix han demostrado un alto nivel de endemicidad. Sporothrix globosa es la única especie patógena descrita hasta la fecha en China, donde es endémica en las provincias del nordeste. Objetivos. Se describen dos casos de esporotricosis linfocutánea con diabetes mellitus como enfermedad de base, en pacientes procedentes de un área no endémica de China. Métodos. Un campesino de 59 años y un jardinero de 60 años de edad fueron atendidos en febrero y junio de 2014, respectivamente. Ambos eran varones, diestros y se presentaron con placas y nódulos de varios años de evolución, que afectaban a la zona superior del brazo derecho. Se tomaron biopsias de los granulomas de la piel, que fueron cultivados en medio de Sabouraud, y se realizó una identificación molecular basada en la región de la calmodulina. Se evaluó la sensibilidad a los antifúngicos mediante el método de microdilución. Resultados. La biopsia de las lesiones mostró la presencia de un granuloma infeccioso. Los hongos aislados en los cultivos fueron identificados como Sporothrix globosa por métodos convencionales, y confirmados mediante identificación molecular. La subsecuente administración de terapia antifúngica oral con bajas dosis de itraconazol fue bien tolerada y resolvió la infección. Conclusiones. La identificación de las especies fúngicas y el análisis de la sensibilidad a los antifúngicos son necesarios por razones epidemiológicas y terapéuticas. El diagnóstico temprano de la esporotricosis es esencial para prevenir las secuelas que genera el progreso de la enfermedad y para ofrecer métodos altamente efectivos para el tratamiento de esta enfermedad emergente. Debe recomendarse evitar la exposición a material vegetal potencialmente contaminado con esporas fúngicas, especialmente en pacientes inmunocomprometidos (AU)