Sporothrix Spp Research Articles

Feline sporotrichosis: occurrence in the Metropolitan Region of the municipality of Recife and anatomical distribution of lesions on cats’ body

Sporotrichosis is a fungal infection of worldwide importance caused by a saprophyte fungus of the genus Sporothrix. The objective of this study was to report the occurrence of feline sporotrichosis in the Metropolitan Region of the city of Recife and the anatomical distribution of lesions on the bodies of cats. Stray cats (n=100) with suggestive sporotrichosis lesions were examined. Material from the lesion was collected from all patients using an imprint and/or sterile swab for cytology. The material was placed on a glass slide and stained using rapid panoptic staining. Data on the localization of each skin lesion were recorded, and cigar-shaped yeast structures were searched at the microscopical examination. A heat map graphically represented the anatomical distribution of each lesion. In 70% (70/100) of samples were visualized fungal structure compatible with Sporothrix spp. There was a predominance of positive males (?2=6.691; p=0.0097). Skin lesions related to Sporothrix spp. infections were unevenly distributed throughout the animal's body. Based on the interpolation of data, the chance for detecting lesions was higher on the nose, anterior paw, and ears than on other body sites (H=40.0043; p=0.0000). Because of the high positivity in these cats, measures of health vigilance are imperative to reduce the risk of animal and human infection.

Geospatialization of Sporothrix spp: spreading knowledge in scientific circles

The Sporothrix fungus exists in different species, S. schenckii, S. americana, S. luriei , S. globosa, S. chilensis, S. pallida and S. brasiliensis . It can be found in soil and putrefactive plant matter, occurring in tropical and subtropical regions. It is observed that infections are the result of the traumatic inoculation of fungal filaments through the skin, affecting humans who have contact with contaminated soil, plants and domestic animals. It is recorded that occurrences of contamination of people with domestic animals have been increasing in Brazil, in the southeast and south regions. The objective was to reflect on research carried out in Brazilian universities, optionally considering doctoral theses studies. State of the Art and Bibliographic methods were used, considering ten years, with a time frame from 2011 to 2020, using two databases, CAPES and BDBTD platforms. For data analysis, statistical analysis was used through ANOVA and geospatialization with the QGIS software, version QGIS-OSGeo4W-3.24.0-2 (18.02.2022). With the results, 49 productions (theses) were retrieved, 18 from the CAPES platform and 31 from the BDBTD platform and analyzed from the perspective of the thematic axes public health, infection and treatment, pharmacology and veterinary sciences. It is concluded that there are records showing the presence of the fungus in the country since the 1980s, and the treatment of the disease caused by the pathogen is possible and dependent on therapy and effective drugs, considering that it is a zoonosis and, therefore, public health issue in some states by mandatory notification.

Zoonotic Sporotrichosis outbreak: Emerging public health threat in the Amazon State, Brazil.

Sporotrichosis is the most common subcutaneous mycosis caused by Sporothrix spp. Traditionally, it is transmitted through injuries involving plant debris. However, over the past few decades, there has been an epidemic increase in human cases resulting from contact with infected animals, particularly cats, in various regions of Brazil. In this report, we report a notable increase in both human and animal cases within the Brazilian Amazon state. An ecological study was conducted by analyzing official records of human and animal sporotrichosis diagnosed in the state of Amazon from 2020 to 2023. Data including patient demographics, clinical manifestations, mycological examination results, and species identification through PCR confirmation were evaluated. During this period, a total of 950 human cases and 2,823 animal cases of sporotrichosis were reported at an exponential rate, since no human cases were registered in 2020. The spatial and temporal dispersion of human sporotrichosis followed that of animal cases, moving from downtown areas to the periphery. Contact with infected animals was reported in 77.7% of cases, with cats being the most commonly implicated (73.5%). Only 66.7% of individuals underwent mycological examination. Among the positive cultures for Sporothrix spp., 65.4% were identified as S. brasiliensis. All patients were treated with systemic antifungals. This study highlights a rising incidence of sporotrichosis among animals and humans in the Brazilian Amazon region over the past four years, with S. brasiliensis being the predominant agent. Collaborative efforts involving healthcare professionals, veterinarians, and public health authorities are crucial to implement effective control measures, educate populations at risk, and promote responsible guidance for pet guardians. These measures are essential to mitigate the burden of epidemic sporotrichosis in Brazil.

Emergence of zoonotic sporotrichosis in Brazil: a genomic epidemiology study

Zoonotic sporotrichosis is a neglected fungal disease, whereby outbreaks are primarily driven by Sporothrix brasiliensis and linked to cat-to-human transmission. To understand the emergence and spread of sporotrichosis in Brazil, the epicentre of the current epidemic in South America, we aimed to conduct whole-genome sequencing (WGS) to describe the genomic epidemiology. In this genomic epidemiology study, we included Sporothrix spp isolates from sporotrichosis cases from Brazil, Colombia, and the USA. We conducted WGS using Illumina NovaSeq on isolates collected by three laboratories in Brazil from humans and cats with sporotrichosis between 2013 and 2022. All isolates that were confirmed to be Sporothrix genus by internal transcribed spacer or beta-tubulin PCR sequencing were included in this study. We downloaded eight Sporothrix genome sequences from the National Center for Biotechnology Information (six from Brazil, two from Colombia). Three Sporothrix spp genome sequences from the USA were generated by the US Centers for Disease Control and Prevention as part of this study. We did phylogenetic analyses and correlated geographical and temporal case distribution with genotypic features of Sporothrix spp isolates. 72 Sporothrix spp isolates from 55 human and 17 animal sporotrichosis cases were included: 67 (93%) were from Brazil, two (3%) from Colombia, and three (4%) from the USA. Cases spanned from 1999 to 2022. Most (61[85%]) isolates were S brasiliensis, and all were reported from Brazil. Ten (14%) were Sporothrix schenckii and were reported from Brazil, USA, and Colombia. For S schenckii isolates, two distinct clades were observed wherein isolates clustered by geography. For S brasiliensis isolates, five clades separated by more than 100 000 single-nucleotide polymorphisms were observed. Among the five S brasiliensis clades, clades A and C contained isolates from both human and cat cases, and clade A contained isolates from six different states in Brazil. Compared with Sbrasiliensis isolates, larger genetic diversity was observed among S schenckii isolates from animal and human cases within a clade. Our results suggest that the ongoing epidemic driven by S brasiliensis in Brazil represents several, independent emergence events followed by animal-to-animal and animal-to human transmission within and between Brazilian states. These results describe how S brasiliensis can emerge and spread within a country. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil; the São Paulo Research Foundation; Productivity in Research fellowships by the National Council for Scientific and Technological Development, and Ministry of Science and Technology of Brazil.

Sporotrichosis in Argentina: clinical and epidemiological analysis

Sporotrichosis is an implantation mycosis caused by Sporothrix spp. It is distributed worldwide and can be found in vegetation and soil. The most frequent route of infection is by trauma with elements contaminated with fungal propagules. Since domestic cats are the most affected animals and can transmit this infection to humans, sporotrichosis is considered a zoonosis. Clinical presentations include nodular lymphangitis, fixed cutaneous, pulmonary (rare), and disseminated (exceptional). To analyze the epidemiology of sporotrichosis in Argentina during 2010 and 2022. To describe the clinical presentation, diagnostic methods, and treatment of cases diagnosed during this period. To know the circulating genotypes and to observe possible associations with the geographic location where the infection was acquired. Analytical, retrospective, and observational study. We analyzed the medical records of patients with sporotrichosis from 12 health institutions in Argentina, between 2010 and 2022. We present 54 cases in which the most frequent clinical form was nodular lymphangitis, and the treatment of choice was itraconazole. Conventional diagnosis was made in all cases. Culture of clinical samples was more sensitive than direct examination because it allowed the isolation of Sporothrix spp. in all 54 cases. Molecular identification was performed in 22 cases, with Sporothrix schenkii sensu stricto being the most frequently isolated species. This study allowed to know the epidemiology of this mycosis in Argentina, as well as the availability of diagnostic methods and the treatment of choice.

Phenotypic and genotypic characterization of Colombian clinical isolates of Sporothrix spp.

For over a century, Sporothrix schenckii was considered the sole species responsible for sporotrichosis. In 2007, scientific community confirmed the disease could be caused by various Sporothrix species. These species differed in their virulence factors and their antifungal sensitivity. This study aims to characterize 42 Colombian clinical isolates of Sporothrix spp. phenotypically and genotypically. Forty-two clinical isolates were characterized using phenotypic methods. It involved various culture media to determine their growth range at different temperatures and to assess the type and distribution of pigment and colony texture. Microscopic morphology was evaluated through microcultures, as well as the conidia diameter, type of sporulation, and morphology. Additionally, the assimilation of carbohydrates was selected as a physiological trait for species identification. Genotyping of 40 isolates was performed through partial amplification of the calmodulin gene, followed by sequence analysis. Molecular studies enabled the identification of 32 isolates of S. schenckii and 8 isolates of S. globosa. The combination of phenotypic and genotypic methods eased these species characterizations and the recognition keys development based on parameters such as growth diameter at 25 and 30 ºC, colony texture (membranous or velvety) on potato dextrose agar, and microscopic morphology with predominance of pigmented triangular, elongated oval globose, or subglobose conidia. Confirmation of the phenotypic characteristics and molecular analysis is crucial for identifying Sporothrix species and determining adequate treatment. This study represents the first phenotypical and genotypical characterization of clinical isolates of Sporothrix spp. reported in Colombia.

Human and Feline Sporotrichosis in a Reference Center of Southeastern Brazil: Genetic Differentiation, Diversity, and Antifungal Susceptibility of Sporothrix Species.

Sporotrichosis is a neglected subcutaneous fungal infection that affects humans and animals worldwide caused by species belonging to the genus Sporothrix. This study aims to examine the range of genetic variations, assess molecular epidemiology significance, and explore potential modes of transmission of the Sporothrix species associated with the current sporotrichosis outbreaks in Espírito Santo, Brazil. In this investigation, 262 samples were evaluated, including 142 from humans and 120 from felines, collected between 2016 and 2021. The isolates were identified based on morphological and molecular characteristics. Sexual idiomorphs were determined by mating-type PCR using primers specific to the MAT1-1 and MAT1-2 loci. Amplified fragment length polymorphism (AFLP) was employed to assess the genetic variability of Sporothrix spp. Finally, antifungal susceptibility testing was performed following the CLSI M38-A2 protocol. Of the 142 human samples, 125 were identified as S. brasiliensis and 17 as S. schenckii s. str. The presence of S. brasiliensis was overwhelming (100%) during outbreaks, highlighting the significant role of domestic cats in the emergence of this species. Heterothallism was the only observed mating strategy. However, the MAT1-2 idiomorph was predominant in cases of cat-transmitted sporotrichosis (χ2 = 202.976; p < 0.0001). Our AFLP results show significant intraspecific variability observed among S. brasiliensis isolates in Espírito Santo. Different genotypes forming subgroups within the same population suggest that these isolates do not originate from a single ancestor, indicating multiple emergences. Furthermore, terbinafine was the antifungal with the best results in vitro. However, in clinical practice, itraconazole remains the primary treatment choice. Sporotrichosis continues to advance in the state; therefore, the health system must outline one-health strategies to contain the disease to prevent future epidemics.

The State of the Art in Transcriptomics and Proteomics of Clinically Relevant Sporothrix Species.

Proteomics provide a robust approach to profile and quantify proteins within cells, organs, or tissues, providing comprehensive insights about the dynamics of cellular processes, modifications, and interactions. Similarly, understanding the transcriptome is essential to decipher functional elements of the genome, unraveling the mechanisms of disease development and the molecular constituents of cells and tissues. Some thermodimorphic fungi of the genus Sporothrix cause sporotrichosis, a subcutaneous mycosis of worldwide relevance. The transcriptome and proteome of the main Sporothrix species of clinical interest can elucidate the mechanisms underlying pathogenesis and host interactions. Studies of these techniques can contribute to the advancement of novel diagnostic and therapeutic strategies. A literature review was carried out, addressing all articles based on proteomics using mass spectrometry and transcriptomics of Sporothrix spp. Twenty-one studies were eligible for this review. The main findings include proteins and genes involved in dimorphism, cell differentiation, thermotolerance, virulence, immune evasion, metabolism, cell adhesion, cell transport, and biosynthesis. With the spread and emergence of sporotrichosis in different countries, ongoing research efforts and new discoveries are welcome to advance knowledge about this mycosis and its agents.

Differential Recognition of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa by Human Monocyte-Derived Macrophages and Dendritic Cells.

Sporotrichosis is a mycosis frequently caused by Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa. The cell wall is a species-specific fungal structure with a direct role in activating the host's immune response. The current knowledge about anti-Sporothrix immunity comes from studies using S. schenckii or S. brasiliensis and murine cells. Macrophages and dendritic cells detect and eliminate pathogens, and although the function of these cells links innate with adaptive immunity, little is known about their interaction with Sporothrix spp. S. schenckii, S. brasiliensis, and S. globosa conidia or yeast-like cells were co-incubated with human monocyte-derived macrophages or dendritic cells, and the phagocytosis and cytokine stimulation were assessed. These interactions were also performed in the presence of specific blocking agents of immune receptors or fungal cells with altered walls to analyze the contribution of these molecules to the immune cell-fungus interaction. Both types of immune cells phagocytosed S. globosa conidia and yeast-like cells to a greater extent, followed by S. brasiliensis and S. schenckii. Furthermore, when the wall internal components were exposed, the phagocytosis level increased for S. schenckii and S. brasiliensis, in contrast to S. globosa. Thus, the cell wall components have different functions during the interaction with macrophages and dendritic cells. S. globosa stimulated an increased proinflammatory response when compared to the other species. In macrophages, this was a dectin-1-, mannose receptor-, and TLR2-dependent response, but dectin-1- and TLR2-dependent stimulation in dendritic cells. For S. schenckii and S. brasiliensis, cytokine production was dependent on the activation of TLR4, CR3, and DC-SIGN. The results of this study indicate that these species are recognized by immune cells differently and that this may depend on both the structure and cell wall organization of the different morphologies.

Inclusion complex of O-allyl-lawsone with 2-hydroxypropyl-β-cyclodextrin: Preparation, physical characterization, antiparasitic and antifungal activity.

The subclass naphthoquinone represents a substance group containing several compounds with important activities against various pathogenic microorganisms. Accordingly, we evaluated O-allyl-lawsone (OAL) antiparasitic and antifungal activity free and encapsulated in 2-hydroxypropyl-β-cyclodextrin (OAL MKN) against Trypanosoma cruzi and Sporothrix spp. OAL and OAL MKN were synthesized and characterized by physicochemical methods. The IC50 values of OAL against T. cruzi were 2.4 µM and 96.8 µM, considering epimastigotes and trypomastigotes, respectively. At the same time, OAL MKN exhibited a lower IC50 value (0.5 µM) for both trypanosome forms and low toxicity for mammalian cells. Additionally, the encapsulation showed a selectivity index approximately 240 times higher than that of benznidazole. Regarding antifungal activity, OAL and OAL MKN inhibited Sporothrix brasiliensis growth at 16 µM, while Sporothrix schenckii was inhibited at 32 µM. OAL MKN also exhibited higher selectivity toward fungus than mammalian cells. In conclusion, we described the encapsulation of O-allyl-lawsone in 2-hydroxypropyl-β-cyclodextrin, increasing the antiparasitic activity compared with the free form and reducing the cytotoxicity and increasing the selectivity towardSporothrix yeasts and the T. cruzi trypomastigote form. This study highlights the potential development of this inclusion complex as an antiparasitic and antifungal agent to treat neglected diseases.

Evaluation of Five Non-Culture-Based Methods for the Diagnosis of Meningeal Sporotrichosis

Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture's limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.

Selective depletion of regulatory T cells enhances the immunogenicity of a recombinant-based vaccine against Sporothrix spp.

Regulatory T cells (Tregs) have been shown to limit the protective immune response against pathogenic species of the fungus Sporothrix spp, the causal agent of sporotrichosis. However, the specific function of Tregs during vaccination against these fungi is known. We evaluated the effect of Tregs depletion on the immunogenicity of an experimental recombinant anti-Sporothrix vaccine, using the DEREG mice. In this model, only Foxp3(+) Tregs express eGFP and diphtheria toxin (DT) receptors, and transient Tregs depletion is achieved by DT administration. Tregs depletion enhanced the frequency of specific IFNγ+ T cells (Th1 lymphocytes) and cytokine production after either the first or second vaccine dose. However, depletion of Tregs during the second dose caused greater stimulation of specific Th1 lymphocytes than depletion during the first dose. Similarly, the highest production of IgG, IgG1, and IgG2a anti rSsEno antibody was detected after Tregs depletion during boost immunization compared to the other immunized groups. Importantly, vaccine immunogenicity improvement after Tregs depletion also had an impact on the more efficient reduction of fungal load in the skin and liver after the challenge with S. brasiliensis in an experimental infection model. Interestingly, the reduction in fungal load was greatest in the Tregs depleted group during boosting. Our results illustrate that Tregs restrict vaccine-induced immune response and their transient depletion could enhance anti-Sporothrix vaccine immunogenicity. Further studies are required to elucidate whether Tregs depletion may be a way to improve the efficacy of vaccination against Sporothrix spp.

Sporotrichosis Outbreak Due to Sporothrix brasiliensis in Domestic Cats in Magallanes, Chile: A One-Health-Approach Study

To describe a S. brasiliensis outbreak in domestic and feral cats detected in the Magallanes region of southern Chile. Between the months of July and September 2022, three cats presented with suppurative subcutaneous lesions located mainly on the head and thoracic limbs. The cytology revealed the presence of yeasts with morphological characteristics suggestive of Sporothrix spp. The histopathology confirmed pyogranulomatous subcutaneous lesions associated with the presence of the same yeasts. The fungal culture followed by the partial gene sequence and analysis of the ITS region confirmed the diagnosis of the S. brasiliensis as the causative agent. The cats were treated with itraconazole associated in one case with potassium iodide. The evolution of the patients was favorable in all cases. An outbreak caused by S. brasiliensis was detected in domestic and feral cats in austral Chile. The correct identification of this fungus and antifungigram is essential for treatment decisions and for designing dissemination control and prevention programs under a one health approach that consider the health of people, animals, and the environment.

P421 Whole-transcriptome analysis of Sporothrix brasiliensis grown in mold- and yeast-inducing conditions

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Sporothrix brasiliensis is an emerging Sporothrix species limited to Brazil capable of causing sporotrichosis in humans and animals, especially in cats. Like other pathogenic Sporothrix species, S. brasiliensis exhibits a temperature-dependent dimorphic switch and is therefore, able to undergo a reversible morphological transition (mold and yeast), in response to environmental thermal stimuli.While dimorphism appears to be essential for virulence in Sporothrix spp, the molecular mechanisms involved in this phenomenon have not yet been fully elucidated.In this study, we used the strand-specific RNA-Seq technique and bioinformatics analysis to investigate the transcriptomic signatures associated with mold and yeast phases of S. brasiliensis. Furthermore, we generated an accurate version of the S. brasiliensis genome annotation in order to perform high-quality gene expression analysis and other functional or structural genomic studies.MethodsThe whole transcriptome of S. brasiliensis ATCC-MYA-4823, grown in both yeast-inducing (YPD medium at 37°C) and mold-inducing (YPD medium at 25°C) conditions, was sequenced in this study. High-quality RNA was used to prepare Illumina TruSeq Stranded mRNA-paired-end sequencing libraries (2 × 150 bp) that were sequenced using the HiSeq-2500 platform. A total of three biological replicates were sequenced for each condition.Before transcriptome assembly, adapters and low-quality reads (Phred-score <25) were removed. The StringTie software was used to assemble the transcriptomes imported into the Apollo webtool to manually curate the genome annotation. Transcripts were investigated using TransDecoder and CPC2 programs to determine whether a gene was potentially protein-coding or non-coding. Finally, differential gene expression analysis between yeast and mold forms of S. brasiliensis was conducted using the edgeR package.ResultsIllumina sequencing resulted in a total of ∼217 million raw reads. After quality filtering and trimming, ∼99.5% of reads were used for downstream bioinformatics analysis. The updated S. brasiliensis genome annotation consisted of a total of 14 664 genes of which 10 243 protein-coding genes, 4259 lncRNAs, 140 tRNAs, and 22 rRNAs.Gene expression analysis revealed a total of 13 838 and 13 938 transcripts expressed in mold- and yeast-form, respectively. Of these, 192 and 292 were expressed exclusively in the mold and yeast-phase, respectively. Moreover, a total of 6802 genes (FDR <0.05) were differentially expressed between the two examined conditions. In particular, 3420 of these genes were up-regulated in the yeast-form (2450 coding, 970 non-coding), and 3382 genes in the filamentous form (2507 coding, 875 non-coding). The raw reads have been deposited into the SRA database and are available under BioProjectID: PRJNA646214.ConclusionsThe characterization of the whole-transcriptome of S. brasiliensis mycelial and yeast-like forms represents an essential starting point for investigating the molecular pathways and regulatory frameworks associated with these two morphological stages. Our results provide new insight into global gene expression profiles of S. brasiliensis, emphasizing the role of non-coding RNAs in its complex transcriptional network.All transcriptomic data have also been integrated into the ‘Sporothrix Genome DataBase’ (www. sporothrixgenomedatabase.unime.it) in order to expand the current knowledge of Sporothrix genomics and to allow a more in-depth structural exploration of S. brasiliensis gene models, including gene expression patterns related to its saprophytic and pathogenic lifestyle.

S10.1d Molecular diagnosis and antifungal susceptibility of Colombian clinical isolates of the Sporothrix spp complex

S10.1 Antifungal dosing in children and adolescents, September 24, 2022, 10:30 AM - 12:00 PM ObjectiveTo detect by chitinase PCR and Species-Specific PCR (SS PCR) the DNA of Sporothrix spp in fresh tissue and Formalin Fixed Paraffin Embedded (FFPE) tissue from patients with sporotrichosis and to evaluate the susceptibility to antifungal of Sporothrix spp isolates obtained from patients diagnosed with sporotrichosis in the Medical Mycology group of the Faculty of Medicine at the University of Antioquia.Materials & MethodsAt the Medical Mycology group service, 26 patients with suspected sporotrichosis were evaluated between 2018 and 2022. Samples of the skin lesions (fresh tissue) were taken for molecular tests and microbiological culture.Regarding the FFPE tissue, 87 samples stored by different histopathological diagnosis centers from 1976 to 2022 were chosen: 45 samples had a histopathological diagnosis of sporotrichosis, and 42 samples of other diseases with involvement in subcutaneous tissue, were used as controls.DNAs previously extracted from fresh tissue and FFPE tissue were used to perform chitinase nested PCR and SS nested PCR. The chitinase nested PCR amplifies a 209 bp fragment for the genus Sporothrix. For species identification, species-specific primers were used (SS PCR), which amplify a 331 bp sequence for S schenckii s. str. and 243 bp for S. globosa of the calmodulin gen. For the SS PCR in tissues, a nested PCR was implemented using the primers CAL1 and CAL2 for the external sequence and the species-specific primers for the internal sequences.The antifungal susceptibility tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 protocol for yeasts. Six antifungals were used: itraconazole, terbinafine, voriconazole, posaconazole, amphotericin B, and fluconazole.ResultsThe culture was positive for Sporothrix spp in 15 (58%) patients and the chitinase nested PCR was positive in 14 of these, with a sensitivity of 93% and a specificity of 91%. In 11 patients, both the culture and the chitinase nested PCR were negative.SS nested PCR was applied to the 14 DNAs with positive chitinase PCR, of which seven were positive for S. schenckii s. str. and one for S. globosa. For the other six samples, the results of this PCR were negative. The results of these PCR were confirmed by the identification of species from the isolates recovered in culture: 13 were identified as S. schenckii s. str and 1 as S. globosa.Of the 45 FFPE tissue samples with histopathological diagnosis of sporotrichosis, the chitinase PCR was positive for 25 (55%) of these and all FFPE tissue samples were negative for SS nested PCR.ConclusionsChitinase nested PCR had a sensitivity of 93% and a specificity of 91% with respect to culture, in samples from fresh tissue. This PCR also has a good performance when it is applied to a good-quality DNA obtained from FFPE tissue, hence, PCR positivity decreased in samples stored for ˃15 years. The results of the nested SS PCR are encouraging, however, it is necessary to improve this test, which we believe is affected by the type of sample used for DNA extraction, so further studies are required.

P390 A lateral flow assay for the immunodiagnosis of human cat-transmitted Sporotrichosis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesSporotrichosis is a neglected endemic implantation (subcutaneous) mycosis caused by different species of Sporothrix spp. Since the 1990s, cat-transmitted sporotrichosis (CTS) by Sporothrix brasiliensis has emerged as an important zoonosis in Brazil, being a public health issue. The disease continues to spread in the country and in neighboring countries. Therefore, the aim of the study is the validation of a fast and accurate test by Lateral Flow Assay (LFA) for serodiagnosis of CTS using human sera.MethodsThe anti-Sporothrix LFA was developed and provided by IMMY (Oklahoma, USA), and obtained via ConA purification of culture filtrate, composed by a 50:50 mix of S. schenkii (ATCC 58251), and S. brasiliensis (ATCC-MYA 4824). The control line was a goat anti-human IgG/IgM. The gold conjugate was a blend of proteins G and L. The strip uses a conjugate pad, nitrocellulose membrane, and an absorbent pad. A prospective cross-sectional study was performed with 300 human sera specimens divided into three groups: Group 1 (n = 100)—patients with a proven or probable diagnosis of CTS based on Guide to Health Surveillance from Ministry of Health Brazil; Group 2 (n = 100)—patients with other mycoses (implantation, systemic or opportunistic infections) confirmed in the laboratory by direct examination and/or culture and/or serological tests; Group 3 (n = 100)—asymptomatic volunteers (Fig. 1). Specimens were collected between November 2018 and March 2021 at the Mycology Laboratory of the Clinical Hospital Complex of the Federal University of Paraná, Curitiba, Brazil. The sera were diluted 1:441 with specimen diluent, 100 μL of the dilution dispensed into a flat bottom well, inserted strip, and incubated at room temperature (15-25°C) for 30 min. A visual read of the strips was done, this read was performed by two operators, within 10 min after the time of incubation. This study was approved by the Research Ethics Committee under registration CAAE 12379819.4.0000.0096. Data analysis was performed using MedCalc software.ResultsUsing the IMMY's anti-Sporothrix detection antibody LFA, we observed a global sensitivity of 83% [95% confidence interval (CI) 74%-90%], a specificity of 82% (95% CI 76%-87%), positive predictive value of 69% (95% CI 62%-75%), negative predictive value of 90% (95% CI 85%-93%), and an accuracy of 82% (77%-86%). The results by clinical form and cross-reactivity analysis can be seen in Table 1.ConclusionThese findings suggest that IMMY's anti-Sporothrix detection antibody LFA prototype is promising, can be a useful diagnostic tool for the diagnosis of CTS in human specimens, and can be used for the qualitative detection of serum antibodies against Sporothrix spp. Like other rapid tests, this LFA is faster and simpler to perform in comparison with other conventional laboratory assays for the diagnosis of sporotrichosis. This test can improve the evidence level of diagnosis of probable disease. Rapid detection of CTS reduces the morbimortality of the disease, and consequently the time of therapy. It could have an impact on the control of the outbreak.

P464 Feline sporotrichosis: an emerging disease in the Brazilian side of the Southern Triple Border

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMSporotrichosis is a neglected tropical disease caused by fungi of the genus Sporothrix spp. Transmission can occur through sapronotic, enzootic, and zoonotic pathways. Sporothrix schenckii and S. brasiliensis are the most common etiologic agents in human and animal diseases in Brazil.ObjectivesThe objective of this work is to alert about the emergence of feline sporotrichosis on the Brazilian side of the Southern Triple Border (Brazil, Paraguay, and Argentina).MethodsFrom July 2021 to March 2022, biological samples and clinical-epidemiological data were collected from 57 domestic cats with typical sporotrichosis lesions and residents of Iguassu Falls (Foz). The cats were selected through notification of the citizens themselves, health agents of Zoonosis Control Center of Iguassu Falls (CCZ–Foz), veterinarians from private veterinary clinics and hospitals, and the receipt of suspected animals by CCZ–Foz. From the samples, direct mycological examinations and fungal culture were performed and a set of 10 fungal isolates from the microbiological tests were selected for molecular analysis based on calmodulin (CAL) gene sequences. From each collection point, the geographic coordinate was taken using the Google Earth® software and the Quantus Gis software (QGIS) was used to assemble the maps.ResultsOf the 57 samples collected, 45 were considered positive in the microbiological tests (Fig. 1), and the first 10 isolates were identified as S. brasiliensis. Based on clinical-epidemiological, and geographic data (Fig. 2), the following problems associated with feline sporotrichosis in this region were identified: the presence of sporotrichosis positive cats in high-density regions population of Foz; the epidemic profile of the disease with cases spreading quickly by neighborhoods in the South region of the city to neighborhoods in the North region; the easy access to the street and other homes of infected cats, which facilitates the zoonotic and enzootic transmission cycle of this disease; the free movement of animals positive for backyards and vacant lots where they can defecate and even come to death the that feeds the sapronotic cycle; and the profile most associated with the animal positive being that of an uncastrated male, not vaccinated and with little or no access to veterinary services.ConclusionFor being a region of the Triple Frontier, there are many differences in political, administrative, and operational between the Public Health Systems of the three countries, with Paraguay and Argentina, the political-administrative-operational centralization is configured as a great node critical for the development of health actions in an adequate way to the needs at a loco-regional level and, above all, on time for the implementation thereof. With the situational diagnosis provided for this study it is possible to understand the factors that influence the emergence of the disease in Iguassu Falls contributing to the development of programs and specific strategies, focusing on raising the population's awareness of safekeeping practices for your animals; free diagnosis for animals (dogs and cats) suspected of sporotrichosis, and treatment with a free supply of medication for patients animals with a confirmed diagnosis of sporotrichosis.

Canine sporotrichosis: Case report

Sporotrichosis is a fungal zoonosis that can affect several species of animals. The present report refers to a 1-year-old female Shih-tzu dog, domiciled, who presented nasal swelling with depigmentation, serosanguineous secretion, sneezing and inspiratory dyspnea. After ruling out the suspicion of neoplasia by histopathology, which showed a result compatible with mild lymphoplasmacytic rhinitis, the patient was referred for consultation with an immunologist. To carry out a differential diagnosis, he went to the São Paulo Zoonoses Surveillance Division, where material was collected from the nostrils using a swab for fungal culture, positive for Sporothrix spp. The dog had no history of contact with animals with sporotrichosis, and the main suspicion of infection was through the soil of a potted plant, located in the residence. Treatment was performed with Itraconazole 10 mg/kg SID for 11 weeks, and continued for another 10 weeks after complete remission of the lesion. After 2 weeks of antifungal suspension, the animal returned with increased nasal volume and dyspnea, requiring resumption of treatment with itraconazole, with total remission of the lesion after 12 weeks. Veterinarians must be aware of the epidemiological context of the region and incorporate sporotrichosis into their routine as a differential diagnosis in cases in which the patient presents skin lesions and follow the patient after treatment is discontinued to assess recurrence.

Beyond Domestic Cats: Environmental Detection of Sporothrix brasiliensis DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil.

In Brazil, sporotrichosis has transitioned from a rural to urban disease, driven by a shift in the initiation of infection from the accidental inoculation of organic matter to the traumatic implantation of the fungus by cats. Since the emergence of zoonotic sporotrichosis caused by Sporothrix brasiliensis, investigations have largely ignored the environmental habitat of the pathogen due to its association with domestic cats. Therefore, we investigated 18 environmental samples collected from rural areas of two cities where zoonotic sporotrichosis is endemic, but where domestic cats are scarce. We utilized traditional culture methods, and samples were also examined with two molecular methods used for the clinical diagnosis of sporotrichosis: a nested-PCR targeting the ITS region and a species-specific PCR targeting the calmodulin gene. No Sporothrix colonies were identified by traditional culture methods. However, the nested-PCR and the species-specific PCR for S. brasiliensis were positive for 18 and 5 samples, respectively. Sequencing revealed that positive results with the nested-PCR were due to non-specific amplification of other Ophiostomatales DNA, rather than Sporothrix spp. Three of the five amplicons from the species-specific PCR were suitable for sequencing and confirmed the presence of S. brasiliensis DNA. Hence, we confirmed that S. brasiliensis, as with other Sporothrix species, has an environmental habitat. Our findings underscore the challenges of nested-PCR for Sporothrix environmental studies and highlight that sequencing must follow PCR protocols to definitively identify Sporothrix spp. in environmental samples.

Differential recognition and cytokine induction by the peptidorhamnomannan from Sporothrix brasiliensis and S. Schenckii

Sporotrichosis is a deep mycosis caused by dimorphic species of the genus Sporothrix, with differences in pathogenicity between S. schenckii and S. brasiliensis species. Recently, it was discovered that the cell wall peptidorhamnomannan (PRM) from S. brasiliensis has additional unknown rhamnose residues. We hypothesize that the structural differences of Sporothrix spp PRMs impact the host's immune response and may explain the severity of sporotrichosis caused by S. brasiliensis. We demonstrate that S. brasiliensis yeasts and its PRM (S.b PRM) induced a strong inflammatory response in human PBMCs, with high production of TNF-α, IL-6 and IL-1β and induction of T-helper cytokines IFN-γ, IL-17 and IL-22. In contrast, S. schenckii yeasts and its PRM induced higher concentrations of interleukin-1 receptor antagonist (IL-1Ra), which resulted in low production of T-helper cytokines such as IL-17 and IL-22. CR3 and dectin-1 were required for cytokine induction by both PRMs, while TLR2 and TLR4 were required for the response of S.s PRM and S.b PRM, respectively. IL-1β and IL-1α production induced by S. brasiliensis yeasts and S.b PRM were dependent on inflammasome and caspase-1 activation. S. schenckii and S.s PRM were able to induce IL-1β independent of ROS. In conclusion, these findings improve our understanding of the pathogenesis of Sporothrix spp. by reporting differences of immunological responses induced by S. schenckii and S. brasiliensis. The study also opens the gateway for novel treatment strategies targeting local inflammation and tissue destruction induced by S. brasiliensis infection through IL-1 inhibition.