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.

Sporotrichosis between 1898 and 2017: The evolution of knowledge on a changeable disease and on emerging etiological agents.

The description of cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. Sporothrix schenckii causes a benign chronic subcutaneous mycosis, Sporothrix brasiliensis is highly virulent, and Sporothrix globosa mainly causes fixed cutaneous lesions. Furthermore, S. brasiliensis is the prevalent species related to cat-transmitted sporotrichosis. Sources of infection, transmission, and distribution patterns also differ between species, and variability differs between species because of different degrees of clonality. The present review article will cover several aspects of the biology of clinically relevant agents of sporotrichosis, including epidemiological aspects of emerging species. Genomic information of Sporothrix spp. is also discussed. The cell wall is an essential structure for cell viability, interaction with the environment, and the host immune cells and contains several macromolecules involved in virulence. Due to its importance, aspects of glycosylation and cell wall polysaccharides are reviewed. Recent genome data and bioinformatics analyses helped to identify specific enzymes of the biosynthetic glycosylation routes, with no homologs in mammalian cells, which can be putative targets for development of antifungal drugs. A diversity of molecular techniques is available for the recognition of the clinically relevant species of Sporothrix. Furthermore, antigens identified as diagnostic markers and putative vaccine candidates are described. Cell-mediated immunity plays a key role in controlling infection, but Sporothrix species differ in their interaction with the host. The adaptive branch of the immune response is essential for appropriate control of infection.

Domestic feline contribution in the transmission of Sporothrix in Rio de Janeiro State, Brazil: a comparison between infected and non-infected populations.

BACKGROUND: Sporotrichosis is a neglected zoonosis caused by pathogenic fungi belonging to the Sporothrix schenckii complex. In Rio de Janeiro state, this disease reached an epidemic status with over 4700 domestic felines and around 4000 humans affected since the mid-90s. The present study evaluated clinical and epidemiological aspects and also the frequency of colonization and infection by these fungi in healthy cats and among those with suspicious cutaneous lesions, inhabiting four Rio de Janeiro state distinct areas. RESULTS: Three hundred and seventy-one cats were included in two groups: 175 healthy cats [CRG] and 196 cats showing lesions suggesting sporotrichosis [SSG]. Mycological diagnosis allowed SSG animals to be divided in positive [104 cats; +SG] and negative [92 cats; -SG] groups. Nails, oral mucosa and lesions swabs were submitted to culture and potential colonies were subculture for micromorphologycal analysis, dimorphism and molecular tests. In the CRG, only one cat was colonized in the oral cavity [0.57%]; in the -SG group, four animals showed colonization of the nail and/or oral cavity [4.3%]; while the highest frequency of colonization [39.4%] was observed in the +SG. All molecularly typed isolates were identified as S. brasiliensis. CONCLUSION: The results obtained here indicate that healthy cats have a minor role in sporotrichosis transmission within the state of Rio de Janeiro. Conversely, a higher participation of diseased feline in sporotrichosis transmission was evidenced, especially by the colonization of their oral cavity. Sporothrix brasiliensis equally affects and colonizes animals from distinct Rio de Janeiro state areas. Thus, we hypothesize that sporotrichosis is a uniform endemic throughout the state, whose transmission depends mainly on the contact with cats with sporotrichosis. Since Rio de Janeiro displays a world unique epidemic model of the disease, not fully understood, data on the infected and non-infected animals can be of major importance for future strategies of sporotrichosis prevention and control. Finally, considering the importance of the current concept of "one health", the experience here observed can be helpful for distinct epizootias and/or zoonosis.

Current progress in the biology of members of the Sporothrix schenckii complex following the genomic era.

Sporotrichosis has been attributed for more than a century to one single etiological agent, Sporothrix schencki. Only eight years ago, it was described that, in fact, the disease is caused by several pathogenic cryptic species. The present review will focus on recent advances to understand the biology and virulence of epidemiologically relevant pathogenic species of the S. schenckii complex. The main subjects covered are the new clinical and epidemiological aspects including diagnostic and therapeutic challenges, the development of molecular tools, the genome database and the perspectives for study of virulence of emerging Sporothrix species.

Amphotericin B, alone or followed by itraconazole therapy, is effective in the control of experimental disseminated sporotrichosis by Sporothrix brasiliensis.

Sporothrix brasiliensis is a highly virulent member of the S. schenckii complex, which is responsible for the emergence of the epidemic sporotrichosis in southeastern Brazil over the last two decades. There are no in vivo studies on the sensitivity of S. brasiliensis to the therapeutic regimens used to treat sporotrichosis. Here, we evaluated the efficacy and safety of antifungal treatments against S. brasiliensis using a murine model of disseminated sporotrichosis. In vitro, S. brasiliensis yeasts were sensitive to low concentrations of amphotericin B-deoxycholate (AMB-d) and itraconazole (ITZ), the latter having greater selectivity toward the fungus. The following treatment regimens were tested in vivo: intravenous AMB-d for 7 days post-infection (p.i.), oral ITZ for up to 30 days p.i., and AMB-d followed by ITZ (AMB-d/ITZ). AMB-d and AMB-d/ITZ led to 100% survival of infected mice at the end of the 45-day experimental period. Although all treatments extended mice survival, only AMB-d and AMB-d/ITZ significantly reduced fungal load in all organs, but AMB-d/ITZ led to a more consistent decrease in overall fungal burden. No treatment increased the levels of serum toxicity biomarkers. Taken together, our results indicate that AMB-d/ITZ is the best therapeutic option for controlling disseminated sporotrichosis caused by S. brasiliensis.

Validation of a serodiagnostic test for sporotrichosis: a follow-up study of patients related to the Rio de Janeiro zoonotic outbreak.

The gold standard for the diagnosis of sporotrichosis consists of the isolation and identification of the fungus from clinical exudates and/or biopsy specimens. However, new technologies offer several advantages over the traditional methods because they are noninvasive and more sensitive in the differential diagnosis of infectious diseases. In the present study, we performed a validation, impact evaluation, and analysis of the applicability of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of sporotrichosis in different groups of patients in comparison with the reference protocols for the evaluation of diagnostic tests for infectious diseases. We used 177 serum samples that were collected between 1998 and 2008 from patients in a geographic area related to the Rio de Janeiro outbreak of sporotrichosis. The ELISA had a low rate of cross-reactivity based on the reference values for diagnostic tests, and the analysis of the receiver operating characteristic curve revealed an area under the curve of 0.9154. Furthermore, higher accuracy was observed in the serodiagnosis of infections in special groups of patients such as pediatric and human immunodeficiency virus-positive patients. We concluded that this ELISA had a good clinical serological correlation and, therefore, can be considered a new diagnostic tool applicable to all clinical presentations of sporotrichosis.

Functional characterization of Sporothrix schenckii glycosidases involved in the N-linked glycosylation pathway.

Protein glycosylation pathways are conserved metabolic processes in eukaryotic organisms and are required for cell fitness. In fungal pathogens, the N-linked glycosylation pathway is indispensable for proper cell wall composition and virulence. In Sporothrix schenckii sensu stricto, the causative agent of sporotrichosis, little is known about this glycosylation pathway. Here, using a genome-wide screening for putative members of the glycosyl hydrolase (CAZy - GH) families 47 and 63, which group enzymes involved in the processing step during N-linked glycan maturation, we found seven homologue genes belonging to family 47 and one to family 63. The eight genes were individually expressed in C. albicans null mutants lacking either MNS1 (for members of family 47) or CWH41 (for the member of family 63). Our results indicate that SsCWH41 is the functional ortholog of CaCWH41, whereas SsMNS1 is the functional ortholog of CaMNS1. The remaining genes of family 47 encode Golgi mannosidases and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like proteins (EDEMs). Since these GH families gather proteins used as target for drugs to control cell growth, identification of these genes could help in the design of antifungals that could be used to treat sporotrichosis and other fungal diseases. In addition, to our knowledge, we are the first to report that Golgi mannosidases and EDEMs are expressed and characterized in yeast cells.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

Immunoproteome of Aspergillus fumigatus using sera of patients with invasive aspergillosis.

Invasive aspergillosis is a life-threatening lung or systemic infection caused by the opportunistic mold Aspergillus fumigatus. The disease affects mainly immunocompromised hosts, and patients with hematological malignances or who have been submitted to stem cell transplantation are at high risk. Despite the current use of Platelia™ Aspergillus as a diagnostic test, the early diagnosis of invasive aspergillosis remains a major challenge in improving the prognosis of the disease. In this study, we used an immunoproteomic approach to identify proteins that could be putative candidates for the early diagnosis of invasive aspergillosis. Antigenic proteins expressed in the first steps of A. fumigatus germination occurring in a human host were revealed using 2-D Western immunoblots with the serum of patients who had previously been classified as probable and proven for invasive aspergillosis. Forty antigenic proteins were identified using mass spectrometry (MS/MS). A BLAST analysis revealed that two of these proteins showed low homology with proteins of either the human host or etiological agents of other invasive fungal infections. To our knowledge, this is the first report describing specific antigenic proteins of A. fumigatus germlings that are recognized by sera of patients with confirmed invasive aspergillosis who were from two separate hospital units.

A neglected disease. Human sporotrichosis in a densely populated urban area in São Paulo, Brazil: clinical-epidemiological and therapeutic aspects.

Human sporotrichosis is caused by different Sporothrix species; however, Sporothrix brasiliensis is the main species, usually related to cat transmission in urban areas. A retrospective descriptive study was conducted at the Institute of Infectology Emílio Ribas from 2010 to 2018. Demography, clinical, diagnostic, and therapeutic data were obtained from medical records. Polymerase chain reaction of the calmodulin gene was performed to identify Sporothrix species. In addition, to evaluate the spread of the disease across São Paulo metropolitan region, TerraView version 4.2.2 software was used for geocoding cases according to residence addresses. Kernell's maps using QGIS software version 2.16.3 were constructed to determine the concentration of cases. Results: 260 cases of sporotrichosis were diagnosed between 2010 and 2018. We observed a 700% increment in the number of human cases in the 2016-2018 triennium compared with the 2013-2015 triennium. Female adults with a median age of 46 years old were the predominant infected group associated with cats' exposition at home care, although the age range of all patients was 01 to 86 years old. The main epidemiological risk of acquiring sporotrichosis was contact with cats, reported by 96.5% of the patients. Molecular identification showed that most of the tested isolates were Sporothrix brasiliensis. Lymphocutaneous form was observed in 59.2% and fixed cutaneous form in 37.5% of the patients. Regarding treatment, itraconazole was the main drug used (94.2%) with a cure rate of 98.8%. We observed an important spread of human sporotrichosis involving cat transmission caused by Sporothrix brasiliensis in a densely populated area of São Paulo state. These results are important to alert clinicians and dermatologists about the occurrence and progression of a neglected tropical disease in an urban area and the urgent necessity to include sporotrichosis as a differential diagnosis in the clinical investigation routine.

Antibody- Based Immunotherapy Combined With Antimycotic Drug TMP- SMX to Treat Infection With Paracoccidioides brasiliensis.

Monoclonal antibodies (mAbs) are promising alternatives to treat infectious diseases, especially given their potential for applications in combination therapies with antimicrobial drugs to enhance the antifungal efficacy. Protection mediated by mAbs used to treat experimental paracoccidioidomycosis (PCM) has been demonstrated previously. Our aim in the present work was to characterize a monoclonal antibody (mAbF1.4) raised against a cell wall glycoconjugate fraction of Paracoccidioides spp. and to analyze its efficacy combined with trimethoprim-sulfamethoxazole (TMP/SMX) as treatment for experimental PCM. We demonstrated that the epitope recognized by mAbF1.4 is consistent with branched glucose residues present on a cell wall ß-glucan polymer. In vitro, mAbF1.4 increased the phagocytic capacity and nitric oxide concentration induced by the macrophage cell line J774.1A, and this resulted in a significant reduction in the viability of the opsonophagocytized yeasts. In vivo, we detected a significant reduction in pulmonary fungal burdens of mice treated with mAbF1.4 in association with TMP/SMX, which correlated with increased pulmonary concentrations (determined by ELISA) of IFN- Î³, TNF-α, IL-10 and IL-17. In parallel, we observed a decrease in IL-4, suggesting that the treatment was associated with a mixed Th1-Th17 type immune response. Histopathology of lung segments from mice receiving the combination therapy showed a significant reduction in granulomas, which were well-defined, and improved maintenance of lung architecture. These findings demonstrate that mAbF1.4 + TMP/SMX therapy is a promising approach to combat PCM as well as decrease disease sequelae and highlights the potential benefits of immune mediators in PCM combined immunotherapy.

Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1ß secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

Comparison of Cell Wall Polysaccharide Composition and Structure Between Strains of Sporothrix schenckii and Sporothrix brasiliensis.

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa. By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-ß-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-ß-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.

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.

L-DOPA accessibility in culture medium increases melanin expression and virulence of Sporothrix schenckii yeast cells.

Melanin is a complex polymer widely distributed in nature and has been described as an important virulence factor in several pathogenic fungi, including Sporothrix schenckii. The aim of the present work was to investigate the presence of melanin on the surface of S. schenckii yeast cells which showed differences in their virulence depending on the culture conditions under which they were grown. Yeast cells were cultivated in brain heart infusion (BHI) broth from Difco and Oxoid. BHI from these two vendors are different in their brain and heart infusion contents. Yeasts cultivated in the medium containing the higher brain infusion content were highly virulent as ascertained by the mice mortality rate, CFU and histopathology. Transmission electron microscopy revealed a higher expression of electron dense granules on the fungal cell wall of the most virulent yeast cells. Flow cytometry analysis, with anti-melanin antibodies, confirmed that this pigment was melanin. Furthermore, spectrophotometric analysis showed a higher concentration of this polymer on NaOH and cell wall extracts of the most virulent yeast cells. These results suggest that differences in the relative content of brain and heart infusion in the culture medium modulated melanin expression on the surface of S. schenckii yeast cells and, as a consequence, virulence. A new pathway of melanin biosynthesis in S. schenckii is proposed which involves the use of phenolic compounds from rich brain medium as melanin substrate.

Cell surface expression of adhesins for fibronectin correlates with virulence in Sporothrix schenckii.

The virulence of four Sporothrix schenckii isolates was compared in a murine model of sporotrichosis, together with the protein pattern of the yeast cell surface and the capacity to bind the extracellular matrix protein fibronectin. Virulence was determined by the mortality rate, fungal burden and histopathology. Two clinical isolates were more virulent for C57BL/6 mice, but no direct correlation was seen between virulence and the clinical or environmental origin of the isolates. The lowest virulence was observed for an isolate recovered from a patient with meningeal sporotrichosis. Although all isolates could effectively disseminate, the dissemination patterns were not similar. Using flow cytometry analysis, we investigated the interaction of all the strains with fibronectin, and showed that the binding capacity correlated with virulence. Western blot analysis of S. schenckii cell wall extracts revealed positive bands for fibronectin in the range of 37-92 kDa. The 70 kDa adhesin was also recognized by a protective monoclonal antibody raised against a gp70 antigen of S. schenckii (mAb P6E7). Confocal microscopy confirmed the co-localization of fibronectin and mAb P6E7 on the yeast cell surface. To our knowledge, this is the first report identifying adhesins for fibronectin on the surface of this human pathogen.

A comparative serological study of the SsCBF antigenic fraction isolated from three Sporothrix schenckii strains.

A previous work showed that an antigenic fraction of Sporothrix schenckii, SsCBF, was specifically recognized by IgG antibodies present in patients' clinical specimens. The objective of this investigation was to compare the reactivity of this antigen recovered from three S. schenckii strains. ROC curve analysis revealed a variation in the sensitivity and specificity of the antigen derived from each of the strains, with a higher AUC for strain 1099-18. Moreover, the presence of the main O-glycosidically-linked epitopes described in the SsCBF fraction was ascertained. A significant reduction in SsCBF reactivity of all the strains was observed after beta-elimination confirming the presence of O-glycan epitopes. The antigen isolated from strain 1099-18 proved to be a more accurate diagnostic tool for serodiagnosis of sporotrichosis.

Group B Streptococcus induces tyrosine phosphorylation of annexin V and glutathione S-transferase in human umbilical vein endothelial cells.

Group B Streptococcus (GBS), a human pathogen that causes infection and invasive diseases in newborns, pregnant women and immunocompromised adults, has been shown to invade human umbilical vein endothelial cells (HUVECs). The objective of this study was to investigate the molecular mechanisms underlying GBS-HUVEC interaction, focusing specifically on the responsiveness of host protein tyrosine kinase (PTK). We found that GBS serotypes III and V induced actin reorganization and formation of stress fibers into HUVECs. Since rearrangements of the actin cytoskeleton into eukaryotic cells are usually associated with the activation of PTK, we decided to follow the expression of this class of kinases in the course of the interaction. Unexpectedly, treatment of HUVECs with genistein greatly increased both cytoadherence and intracellular viability, for all GBS strains studied. GBS increased tyrosine phosphorylation of two proteins with an apparent molecular mass of 35 and 23 kDa in HUVECs as demonstrated by Western blot analysis with anti-phosphotyrosine antibodies. Mass spectra analysis identified these proteins as annexin V and glutathione S-transferase. Studies are in progress to identify the role of these two proteins on GBS-HUVEC interaction.

Ploidy Determination in the Pathogenic Fungus Sporothrix spp.

The pathogenic clade of the Sporothrix genus comprises the etiological agents of sporotrichosis, a worldwide emergent disease. Despite the growing understanding of their successful pathogen traits, there is little information on genome sizes and ploidy within the genus. Therefore, in this work, we evaluated the ploidy of four species of the Sporothrix genus, specifically Sporothrix brasiliensis, Sporothrix schenckii, Sporothrix globosa, and Sporothrix pallida. Through cell cycle analysis of the yeast-phase cells, we showed that the DNA content of G0/G1 cells was similar to the genome size determined by whole genome sequencing. Moreover, ploidy of S. schenckii, S. brasiliensis, and S. pallida that was determined by allele composition using next-generation sequencing (NGS) data is consistent with monomorphic positions at each allele. These data show that the analyzed strains of Sporothrix are haploid, or at least aneuploid, thereby laying the foundation for the development of a molecular toolbox for Sporothrix spp.