Combination therapy of itraconazole and an acylhydrazone derivative (D13) for the treatment of sporotrichosis in cats.

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.

A Randomized Clinical Trial Comparing Itraconazole and a Combination Therapy with Itraconazole and Potassium Iodide for the Treatment of Feline Sporotrichosis.

Feline sporotrichosis is an endemic disease with high occurrence in Brazil. Itraconazole (ITZ) remains the drug of choice for treating this disease in cats, despite the increasing reports of therapeutic failure. A controlled, randomized clinical trial was performed on 166 naive cats with sporotrichosis to assess the effectiveness and safety of the combination therapy with ITZ and potassium iodide (KI) compared with ITZ monotherapy. Cats were randomly allocated into two treatment groups: G1-ITZ 100 mg/cat/day-and G2-ITZ 100 mg/cat/day + KI 2.5-20 mg/kg/day. Cats treated in G2 presented 77% more risk of reaching a clinical cure (a positive effect) than those treated in G1, even when controlled by negative predictors. The survival curves of the two treatment protocols indicate that a clinical cure was achieved faster in G2. An increase in the KI dose was necessary in 28 cats due to the persistence of clinical signs. Adverse reactions were equally frequent in both groups and manageable with a temporary drug suspension and/or a hepatoprotective therapy. The combination therapy was associated with a higher cure rate and a shorter treatment time, suggesting that ITZ+KI arises as a better option for treating feline sporotrichosis and should be considered the first-line treatment, especially in the presence of negative predictors.

In vitro antifungal activity of curcumin mediated by photodynamic therapy on Sporothrix brasiliensis.

BACKGROUND: Sporothrix brasiliensis is a pathogenic dimorphic fungus that affects humans and animals causing sporotrichosis. The treatment of this disease with conventional antifungals commonly results in therapeutic failures and resistance. Therefore, this study aimed to evaluate the in vitro effect of curcumin (CUR) mediated by photodynamic therapy (PDT) in its pure state and incorporated into pharmaceutical formulation in gel form, on the filamentous and yeast forms of S. brasiliensis. METHODS: Cells from both forms of the fungus were treated with pure curcumin (PDT-CUR). For this, CUR concentrations ranging from 0.09 to 50 µM were incubated for 15 min and then irradiated with blue LED at 15 J/cm². Similarly, it was performed with PDT-CUR-gel, at lower concentration with fungistatic action. After, a qualitative and quantitative (colony forming units (CFU)) analysis of the results was performed. Additionally, reactive oxygen species (ROS) were detected by flow cytometry. Results PDT with 0.78 µM of CUR caused a significant reduction (p < 0.05) in cells of the filamentous and yeast form, 1.38 log10 and 1.18 log10, respectively, in comparison with the control. From the concentration of 1.56 µM of CUR, there was a total reduction in the number of CFU (≥ 3 log10). The PDT-CUR-gel, in relation to its base without CUR, presented a significant reduction (p < 0.05) of 0.83 log10 for the filamentous form and for the yeast form, 0.72 log10. ROS release was detected after the PDT-CUR assay, showing that this may be an important pathway of death caused by photoinactivation. Conclusion PDT-CUR has an important in vitro antifungal action against S. brasiliensis strains in both morphologies.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare.

BACKGROUND: Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES: Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS: Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS: The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS: The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare

BACKGROUND Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

Spatial Distribution of Canine Sporotrichosis in Rio de Janeiro, Brazil (1998-2018) and Its Correlation with Socioeconomic Conditions.

Canine sporotrichosis is a poorly described global disease and a spatial approach has not yet been used to assess the disease in dogs. Therefore, this study aimed to describe the occurrence of canine sporotrichosis in the state of Rio de Janeiro, Brazil, from 1998 to 2018 and its correlation with socioeconomic characteristics using exploratory spatial data analysis. A total of 295 cases of canine sporotrichosis were identified and 249 were georeferenced. There was a higher concentration of cases in the municipality of Rio de Janeiro, as well as along the border of the city and the adjacent municipalities in the great metropolitan area. The cases occurred in areas where most of the dwellings are houses. Moreover, no focus of disease density was found in the southern part of Rio de Janeiro city over the period studied, possibly due to better socioeconomic conditions. Areas with a high concentration of canine sporotrichosis cases coincided with regions that possessed a low proportion of households without paving, suggesting that the disease is not necessarily linked to extreme poverty. The mapping of areas with a greater density of cases is fundamental to formulate targeted and strategic plans in order to implement effective public health prevention and control measures.

Global distribution of animal sporotrichosis: A systematic review of Sporothrix sp. identified using molecular tools.

Sporotrichosis is a subcutaneous mycosis caused by thermodimorphic fungi of the genus Sporothrix. The phenotypic and genotypic differences of the isolates within the genus Sporothrix have been associated with their geographic distribution, virulence capacity, or clinical manifestation of sporotrichosis. Therefore, it is crucial to identify the causative agent of sporotrichosis. However, there are few case reports and studies in animals compared to those in humans, despite the substantial increase in the number of cases of sporotrichosis by zoonotic transmission, especially in endemic areas. Considering the epidemiological importance, taxonomic evolution and worldwide distribution of these fungi in the last decade, there is interest in identifying the species of the genus Sporothrix in different regions of the world. This study aimed to analyze the geographic distribution of animal sporotrichosis in the world, caused by pathogenic species identified by use of molecular tools. This systematic review of articles from 2007 to 2021 analyzed the geographic distribution of species that cause sporotrichosis in cats, dogs and other animals. It demonstrated that the most identified species were S. brasiliensis, isolated from cats in Brazil and S. schenckii isolated from cats in Malaysia. We show the lack of studies in global areas and reinforce the need to use molecular tools to identify and monitor potential pathogens.

Sporothrix brasiliensis and Feline Sporotrichosis in the Metropolitan Region of Rio de Janeiro, Brazil (1998-2018).

Feline sporotrichosis is enzootic in different regions of Brazil, especially in Rio de Janeiro. This study compared the genotype profiles of Sporothrix sp. isolated from cats in Rio de Janeiro between 1998 and 2018 and evaluated their association with clinical and epidemiological characteristics. One hundred nineteen Sporothrix sp. isolates from a cohort of cats with sporotrichosis seen at INI/Fiocruz were included. Clinical and epidemiological data were obtained from the medical records of the animals. T3B PCR fingerprinting was used for molecular identification of the Sporothrix species. All isolates were characterized as Sporothrix brasiliensis, with the observation of low intraspecific variation in 31 isolates (31.3%). The interval between lesion onset and first medical visit at INI/Fiocruz, as well as treatment duration until clinical cure, was longer in cats from the first decade of the epizootic. In addition, the frequency of the variables "good general status" and "presence of lymphadenomegaly" was higher among cats whose strains did not exhibit intraspecific variation. So far, S. brasiliensis has been the only species identified in feline cases of sporotrichosis since the beginning of the epizootic in Rio de Janeiro at INI/Fiocruz.

Advances and challenges in the management of feline sporotrichosis.

The domestic cat is the most susceptible host to Sporothrix infection, developing severe clinical forms. Few effective antifungal agents are available for treating feline sporotrichosis, and cases of treatment failure are common. Treatment success depends on cat health status, therapy-related factors, as well as social/economic issues, but it is mainly contingent upon the host-fungus interaction. The owner's adherence is critical and should be reinforced throughout the treatment to increase the chances of a successful outcome. The antifungal agents described for feline sporotrichosis are most often used in monotherapy regimens. Due to cases in which the treatment with itraconazole failed, the use of antifungal agents in combination should be considered to achieve synergy. The combination of itraconazole and potassium iodide represents an important option for the treatment of naïve cats presenting multiple cutaneous lesions, nasal mucosal lesions and/or respiratory signs, as well as for refractory cases. However, the therapeutic options for unsuccessfully treated cases are scarce. Therefore new options are needed, even more taking into account that there are many in vitro potential molecules not available for use in cats yet. More studies are necessary to correlate in vitro antifungal susceptibility tests results and the outcome of cats treated due to sporotrichosis. This review will briefly discuss both the antifungal drugs and treatment protocols used in cats with sporotrichosis, as well as the determinants of treatment failure.

Anti-Sporothrix Antibody Detection in Domestic Cats as an Indicator of a Possible New Occurrence Area for Sporotrichosis in North Brazil.

Feline sporotrichosis has emerged as an important public health issue in some countries, especially Brazil. Currently, zoonotic transmission of Sporothrix brasiliensis by domestic cats is the major sporotrichosis spread form throughout this country. Sporotrichosis in Brazil is a good model for the One Health concept application, which connects the environment, human and animal health. Under this thinking, the aim of this study was to investigate the seroprevalence of sporotrichosis in cats from Rolim de Moura, Rondônia, Brazil, using antibody detection by an ELISA test previously validated for human diagnosis. For the standardization of this test, 30 serum samples from cats with proven sporotrichosis and 11 sera from healthy cats were used. The assay showed 87% sensitivity and 100% specificity for the diagnosis of feline sporotrichosis. After the standardization, 202 serum samples from distinct cats from Rolim de Moura were evaluated. The test was positive in 63 (31.19%) cats from the studied area. A multivariate analysis revealed that living far from forest or agricultural areas as well as pure breed animals had higher odds ratios (3.157 and 2.281, respectively) for the presence of detectable levels of anti-Sporothrix antibodies. These results show the applicability of this assay in the detection of anti-Sporothrix antibodies in feline serum samples and point to a putative new occurrence area of urban sporotrichosis dispersing to the North region of Brazil.

COVID-19 and zoonoses in Brazil: Environmental scan of one health preparedness and response.

The emergence of the COVID-19 pandemic reinforced the central role of the One Health (OH) approach, as a multisectoral and multidisciplinary perspective, to tackle health threats at the human-animal-environment interface. This study assessed Brazilian preparedness and response to COVID-19 and zoonoses with a focus on the OH approach and equity dimensions. We conducted an environmental scan using a protocol developed as part of a multi-country study. The article selection process resulted in 45 documents: 79 files and 112 references on OH; 41 files and 81 references on equity. The OH and equity aspects are poorly represented in the official documents regarding the COVID-19 response, either at the federal and state levels. Brazil has a governance infrastructure that allows for the response to infectious diseases, including zoonoses, as well as the fight against antimicrobial resistance through the OH approach. However, the response to the pandemic did not fully utilize the resources of the Brazilian state, due to the lack of central coordination and articulation among the sectors involved. Brazil is considered an area of high risk for emergence of zoonoses mainly due to climate change, large-scale deforestation and urbanization, high wildlife biodiversity, wide dry frontier, and poor control of wild animals' traffic. Therefore, encouraging existing mechanisms for collaboration across sectors and disciplines, with the inclusion of vulnerable populations, is required for making a multisectoral OH approach successful in the country.

Sporothrix spp. Biofilms Impact in the Zoonotic Transmission Route: Feline Claws Associated Biofilms, Itraconazole Tolerance, and Potential Repurposing for Miltefosine.

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.

Cryptococcus neoformans VNII as the Main Cause of Cryptococcosis in Domestic Cats from Rio de Janeiro, Brazil.

Cryptococcosis is a systemic fungal disease acquired from contaminated environments with propagules of the basidiomycetous yeasts of the Cryptococcus neoformans and C. gattii species complexes. The C. neoformans species complex classically comprises four major molecular types (VNI, VNII, VNIII, and VNIV), and the C. gattii species complex comprises another four (VGI, VGII, VGIII, and VGIV) and the newly identified molecular type VGV. These major molecular types differ in their epidemiological and ecological features, clinical presentations, and therapeutic outcomes. Generally, the most common isolated types are VNI, VGI, and VGII. The epidemiological profile of cryptococcosis in domestic cats is poorly studied and cats can be the sentinels for human infections. Therefore, the present study aimed to determine the molecular characterization of Cryptococcus spp. isolated from domestic cats and their dwellings in the metropolitan area of Rio de Janeiro, Brazil. A total of 36 Cryptococcus spp. strains, both clinical and environmental, from 19 cats were subtyped using multilocus sequence typing (MLST). The ploidy was identified using flow cytometry and the mating type was determined through amplification with specific pheromone primers. All strains were mating type alpha and 6/36 were diploid (all VNII). Most isolates (63.88%) were identified as VNII, a rare molecular type, leading to the consideration that this genotype is more likely related to skin lesions, since there was a high percentage (68.75%) of cats with skin lesions, which is also considered rare. Further studies regarding the molecular epidemiology of cryptococcosis in felines are still needed to clarify the reason for the large proportion of the rare molecular type VNII causing infections in cats.

Frequency of detection and load of amastigotes in the pancreas of Leishmania infantum-seropositive dogs: clinical signs and histological changes.

BACKGROUND: Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and is highly lethal in humans and dogs if left untreated. The frequency of this parasite and associated histological changes in the pancreas of dogs are poorly studied. Therefore, the objectives of this study were to evaluate the frequency of detection and load of amastigotes in the pancreas of L. infantum-seropositive dogs and to identify the clinical signs and histological changes associated with parasitism of this organ. METHODS: One hundred forty-three dogs from an endemic area in Brazil that tested seropositive for L. infantum were studied. The dogs were clinically examined, killed, and necropsied between 2013 and 2014. One fragment of the pancreas was randomly collected for histopathology and immunohistochemistry, and spleen and bone marrow were collected for culture. RESULTS: Leishmania amastigotes were detected in the pancreas of 22 dogs (15.4%) by immunohistochemistry, all exhibiting L. infantum parasitism in the spleen and/or bone marrow. Poor body condition and cachexia were only associated with infection of the pancreas with Leishmania spp. (p = 0.021) and were found in 40.9% of dogs with pancreatic infection. Anorexia, vomiting, and/or diarrhea were observed in 9.2% of dogs with pancreatitis. The median parasite load in the pancreas was 1.4 infected macrophages/mm2. Pancreatic histological changes and their frequencies were: granulomatous pancreatitis (28.0%), lymphoplasmacytic pancreatitis (23.8%), acinar cell degeneration (6.3%), fibrosis (5.6%), hemorrhage (2.1%), eosinophilic pancreatitis (0.7%), suppurative pancreatitis (0.7%), and necrosis (0.7%). CONCLUSIONS: The present results demonstrate that L. infantum is one of the etiological agents of chronic pancreatitis in dogs; however, the frequency of detection and parasite load are low in this organ. The lack of an association of poor body condition and cachexia with pancreatitis and the low frequency of clinical signs commonly associated with pancreatitis suggest that a significant portion of the organ is not affected by this parasite. On the other hand, the association of poor body condition and cachexia with concomitant infection of the pancreas, spleen, and/or bone marrow with this parasite suggests that these manifestations are the result of a more advanced stage of canine visceral leishmaniasis.

Investigation of SARS-CoV-2 infection in dogs and cats of humans diagnosed with COVID-19 in Rio de Janeiro, Brazil.

BACKGROUND: Infection by SARS-CoV-2 in domestic animals has been related to close contact with humans diagnosed with COVID-19. Objectives: To assess the exposure, infection, and persistence by SARS-CoV-2 of dogs and cats living in the same households of humans that tested positive for SARS-CoV-2, and to investigate clinical and laboratory alterations associated with animal infection. METHODS: Animals living with COVID-19 patients were longitudinally followed and had nasopharyngeal/oropharyngeal and rectal swabs collected and tested for SARS-CoV-2. Additionally, blood samples were collected for laboratory analysis, and plaque reduction neutralization test (PRNT90) to investigate specific SARS-CoV-2 antibodies. RESULTS: Between May and October 2020, 39 pets (29 dogs and 10 cats) of 21 patients were investigated. Nine dogs (31%) and four cats (40%) from 10 (47.6%) households were infected with or seropositive for SARS-CoV-2. Animals tested positive from 11 to 51 days after the human index COVID-19 case onset of symptoms. Three dogs tested positive twice within 14, 30, and 31 days apart. SARS-CoV-2 neutralizing antibodies were detected in one dog (3.4%) and two cats (20%). In this study, six out of thirteen animals either infected with or seropositive for SARS-CoV-2 have developed mild but reversible signs of the disease. Using logistic regression analysis, neutering, and sharing bed with the ill owner were associated with pet infection. CONCLUSIONS: The presence and persistence of SARS-CoV-2 infection have been identified in dogs and cats from households with human COVID-19 cases in Rio de Janeiro, Brazil. People with COVID-19 should avoid close contact with their pets during the time of their illness.

Antifungal activity of Acylhydrazone derivatives against Sporothrix spp.

Sporotrichosis is an emerging mycosis caused by members of the genus Sporothrix The disease affects humans and animals, particularly cats, which plays an important role in the zoonotic transmission. Feline sporotrichosis treatment options include itraconazole (ITC), potassium iodide and amphotericin B, drugs usually associated with deleterious adverse reactions and refractoriness in cats, especially when using ITC. Thus, affordable, non-toxic and clinically effective anti-Sporothrix agents are needed. Recently, acylhydrazones (AH), molecules targeting vesicular transport and cell cycle progression, exhibited a potent antifungal activity against several fungal species and displayed low toxicity when compared to the current drugs. In this work, the AH derivatives D13 and SB-AF-1002 were tested against Sporothrix schenckii and Sporothrix brasiliensis Minimal inhibitory concentrations of 0.12 - 1 µg/mL were observed for both species in vitro D13 and SB-AF-1002 showed an additive effect with itraconazole. Treatment with D13 promoted yeast disruption with release of intracellular components, as confirmed by transmission electron microscopy of S. brasiliensis exposed to the AH derivatives. AH-treated cells displayed thickening of the cell wall, discontinuity of the cell membrane and an intense cytoplasmic degeneration. In a murine model of sporotrichosis, treatment with AH derivatives was more efficient than ITC, the drug of choice for sporotrichosis. The results of the preliminary clinical study in cats indicate that D13 is safe and has potential to become a therapeutic option for sporotrichosis when associated to ITC. Our results expand the antifungal broadness of AH derivatives and suggest that these drugs could be exploited to combat sporotrichosis.

Genetic and histopathological characterization of Toxoplasma gondii genotypes isolated from free-range chickens reared in the metropolitan region of Rio de Janeiro state, Brazil.

This study aimed to genetically characterize Toxoplasma gondii isolates obtained from free-range chickens reared in the metropolitan region of the state of Rio de Janeiro, Brazil, and to evaluate the morbidity and histological changes associated with these isolates in mice. A mouse bioassay was used to isolate T. gondii from a pool of tissue samples (brain, heart, and thigh muscles) collected from 163 chickens. The 36 isolates obtained were genetically characterized by restriction fragment polymorphism (PCR-RFLP) analysis of the SAG1, 5'-3'SAG2, aSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3 genomic regions. Seventeen atypical genotypes were identified and nine of them were reported for the first time. All identified genotypes caused clinical signs and histological changes in mice, with the majority being associated with high cumulative morbidity (65%) and severe or very severe histological changes (76%). The exclusive identification of atypical genotypes, with a predominance of new genotypes, indicates great genetic diversity of T. gondii in the region studied. In addition, the finding that all identified genotypes caused clinical signs and often severe histological changes in mice suggests potentially relevant virulence of these strains.

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols.

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 µm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 µm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

Canine sporotrichosis: polyphasic taxonomy and antifungal susceptibility profiles of Sporothrix species in an endemic area in Brazil.

Sporotrichosis, a mycosis caused by pathogenic species of the genus Sporothrix, affects diverse species of mammals. Until 2007, Sporothrix schenckii was considered the unique etiologic agent of sporotrichosis. Canine sporotrichosis is a poorly reported disease, and the majority of cases are from Rio de Janeiro, Brazil. There are scarce studies on the characterization of canine isolates of Sporothrix schenckii complex, as well as few antifungal susceptibility data available. The aim of this study was to characterize the clinical isolates of Sporothrix from dogs from Brazil at species level and evaluate their antifungal susceptibility profile. Polyphasic taxonomy was used to characterization at species level (morphological, phenotypical characteristics, and molecular identification). Antifungal susceptibility profiles (amphotericin B, itraconazole, ketoconazole, posaconazole, and terbinafine) were determined using the Clinical and Laboratory Standards Institute broth microdilution method (M38-A2). According to phenotypic identification and molecular analysis, 46 isolates included in this study were identified as S. brasiliensis and one as S. schenckii. Amphotericin B presented the highest minimum inhibitory concentration values, and the other drugs showed effective in vitro antifungal activity. This is the first report of S. schenckii in dogs from Brazil, since S. brasiliensis is the only species that has been described in canine isolates from Rio de Janeiro to date. Nevertheless, no differences were observed in the antifungal susceptibility profiles between the S. brasiliensis and S. schenckii isolates, and it is important to continuously study new canine clinical isolates from Rio de Janeiro, Brazil.

Guideline for the management of feline sporotrichosis caused by Sporothrix brasiliensis and literature revision.

We herein present a Brazilian guideline for the management of feline sporotrichosis, a mycosis caused by Sporothrix brasiliensis. This guideline is an effort of a national technical group organized by the Working Group on Sporothrix and Sporotrichosis of the International Society for Human and Animal Mycology (ISHAM). This publication intends to provide information on clinical-epidemiological aspects of this zoonosis, as well as a literature revision. Moreover, it gives some practical information on diagnosis and treatment of feline sporotrichosis. It also contains information that can be helpful for the prevention and control of S. brasiliensis transmission.