Sporothrix Schenckii Sensu Stricto Research Articles

The Mating Type (MAT) and Virulence of Sporothrix schenckii sensu stricto Isolates Maintained in Culture Collection.

In an attempt to determine the mating type of different Sporothrix schenckii sensu stricto isolates that remained viable after a long period of preservation in a culture collection and to correlate them with the degree of virulence/pathogenicity, a PCR technique using primers designed for the sequences of MAT1-1-1 and MAT1-2-1 genes and a murine experimental model were used. The results showed that there was no correlation between the mating type and virulence among the isolates. Furthermore, different degrees of virulence/pathogenicity, ranging from high to low, were found among them based on different virulence parameters. It was assumed that the long period of preservation favored the changes, yielding the isolation of variants. Thus, we believe that new technologies for studies on factors can improve our knowledge of the pathogenesis of sporotrichosis.

Phenotypic and Genotypic Characterization and Antifungal Susceptibility of Sporothrix schenckii sensu stricto Isolated from a Feline Sporotrichosis Outbreak in Bangkok, Thailand.

Sporotrichosis, an invasive fungal infection caused by Sporothrix schenckii, has emerged in Southeast Asia, affecting cats and posing a potential zoonotic risk to humans. We evaluated 38 feline sporotrichosis cases in and around Bangkok, Thailand, from 2017 to 2021. The isolates were phenotypically and genotypically characterized. The cats infected with sporotrichosis were mainly young adults, males, and domestic short hairs with uncontrolled outdoor access, and they lived in Bangkok. All isolates showed low thermotolerance and converted to the yeast phase at 35 °C. Based on the internal transcribed spacer region of rDNA sequences, our strains belonged to S. schenckii sensu stricto and clustered with clinical clade D. Based on the concatenated tree of calmodulin and beta-tubulin genes, five groups of S. schenckii were generated, and the monophyletic clade, Group II, of Thai strains was recognized. In vitro antifungal susceptibility testing demonstrated that the MIC50 of our isolates to amphotericin B, itraconazole, and posaconazole were within the limit of the species-specific epidemiological cutoff values, suggesting that the organisms were the wild type. Addressing the outbreak of feline sporotrichosis in Thailand by providing guidelines for diagnosis and effective treatment may help control the spread of disease and reduce the risk of cat-transmitted sporotrichosis to humans.

Phenotypic and Genotypic Characterization and Antifungal Susceptibility of Sporothrix Schenckii Sensu Stricto Isolated from a Feline Sporotrichosis Outbreak in Bangkok, Thailand

Phenotypic and Genotypic Characterization and Antifungal Susceptibility of Sporothrix Schenckii Sensu Stricto Isolated from a Feline Sporotrichosis Outbreak in Bangkok, Thailand

Phenotypic and Genotypic Characterization and Antifungal Susceptibility of Sporothrix Schenckii Sensu Stricto Isolated from a Feline Sporotrichosis Outbreak in Bangkok, Thailand

Phenotypic and Genotypic Characterization and Antifungal Susceptibility of Sporothrix Schenckii Sensu Stricto Isolated from a Feline Sporotrichosis Outbreak in Bangkok, Thailand

Feline sporotrichosis caused by Sporothrix schenckii sensu stricto in Southern Thailand: phenotypic characterization, molecular identification, and antifungal susceptibility.

Feline sporotrichosis caused by the Sporothrix schenckii complex is a global subcutaneous mycosis, having higher prevalence in Latin America and Malaysia. However, its etiological agents have not been elucidated in Thailand, a neighboring country of Malaysia, where the cases are increasing. This study identified 38 feline isolates of S. schenckii from Southern Thailand, collected between 2018 and 2021, using phenotypic characterization and molecular identification using polymerase chain reaction (PCR)-sequencing of partial calmodulin (CAL) and ß-tubulin (Bt2) genes. Phenotypic characteristics proved that the isolates were S. schenckii sensu lato, with low thermotolerance. Based on partial CAL and Bt2-PCR sequencing, all isolates were identified as S. schenckii sensu stricto. Phylogenetic analyses revealed that the isolates were clustered with S. schenckii sensu stricto isolated from the cats in Malaysia. A low degree of genetic diversity was observed among the Thai feline isolates. The antifungal susceptibility of these isolates to antifungal agents, including itraconazole (ITC), ketoconazole (KTC), fluconazole (FLC), and amphotericin B (AMB), was investigated according to the M27-A3 protocol of the Clinical and Laboratory Standards Institute. Results showed low ITC, KTC, and AMB activities against S. schenckii sensu stricto isolates, with high minimum inhibitory concentration (MIC) ranges of 1-8, 1-8, and 2-16μg/ml, respectively, whereas FLC exhibited MICs of 64 and>64μg/ml. This study indicated that S. schenckii sensu stricto is the causative agent responsible for feline sporotrichosis in Southern Thailand. Their phenotypic characteristics and in vitro antifungal susceptibility profiles will help to improve our understanding of this mycosis in Thailand.

Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil.

Sporotrichosis is a subcutaneous mycosis worldwide distributed reaching hyperendemic proportions in Brazil. Many isolates from patients with sporotrichosis are preserved in culture collections by different methods around the world. The preservation methods are used to maintain the viability and the morphophysiological and genetic characteristics of isolates for long periods. In this study, we evaluated 34 isolates, previously, identified as S. schenckii by a classical identification method, initially preserved by periodical subcultures and then under mineral oil at culture collection of Oswaldo Cruz Institute/Fiocruz, to re-identify them by polyphasic identification. Our results showed that seven isolates remained viable for 34 to 64 years under oil, one isolate lost the ability to sporulate which was reverted by using a medium culture supplemented with rosebush branches and all of them were identified as Sporothrix schenckii sensu stricto by morphological, physiological, partial β-tubulin gene sequencing and phylogenetic analysis.

Chitin-rich heteroglycan from Sporothrix schenckii sensu stricto potentiates fungal clearance in a mouse model of sporotrichosis and promotes macrophages phagocytosis

BackgroundFungal cell wall polysaccharides maintain the integrity of fungi and interact with host immune cells. The immunomodulation of fungal polysaccharides has been demonstrated in previous studies. However, the effect of chitin-rich heteroglycan extracted from Sporothrix schenckiisensu stricto on the immune response has not been investigated.ResultsIn this study, chitin-rich heteroglycan was extracted from S. schenckiisensu stricto, and immunomodulation was investigated via histopathological analysis of skin lesions in a mouse model of sporotrichosis and evaluation of the phagocytic function and cytokine secretion of macrophages in vitro. The results showed that the skin lesions regressed and granulomatous inflammation was reduced in infected mice within 5 weeks. Moreover, heteroglycan promoted the fungal phagocytosis by macrophages and modulated the cytokine secretion. Heteroglycan upregulated TNF-α expression early at 24 h and IL-12 expression late at 72 h after incubation, which might result from moderate activation of macrophages and contribute to the subsequent adaptive immune response.ConclusionsChitin-rich heteroglycan extracted from S. schenckiisensu stricto potentiated fungal clearance in a mouse model of sporotrichosis. Moreover, chitin-rich heteroglycan promoted fungus phagocytosis by macrophages and modulated cytokines secretion. These results might indicate that chitin-rich heteroglycan could be considered as an immunomodulator used in the treatment of sporotrichosis.

Antifungal activity of deferiprone and EDTA against Sporothrix spp.: Effect on planktonic growth and biofilm formation.

Our data reveal that iron chelators deferiprone and ethylenediaminetetraacetic acid reduced the growth of planktonic cells of Sporothrix spp. as well as had synergistic interaction with antifungal drugs against this pathogen and reduced biofilm formation of Sporothrix spp.

Feline sporotrichosis in Asia.

Sporothrix schenckii sensu lato is currently recognized as a species complex with only Sporothrix brasiliensis, Sporothrix schenckii sensu stricto, Sporothrix globosa and Sporothrix pallida identified to cause disease in the cat. Feline sporotrichosis in Asia is mainly reported from Malaysia where a single clonal strain of clinical clade D, Sporothrix schenckii sensu stricto manifesting low susceptibility to major antifungal classes, has been identified as the agent of the disease. Sporothrix globosa has been identified to cause disease from a single cat in Japan while the specific species of agent has not been identified yet for the disease in Thailand. Despite efforts to elucidate and describe the pathogenicity of the agent and the disease it causes, the paucity of data highlights the need for further molecular epidemiological studies to characterize this fungus and the disease it causes in Asia. Its prognosis remains guarded to poor due to issues pertaining to cost, protracted treatment course, zoonotic potential and low susceptibility of some strains to antifungals.

Identification of proteins in Sporothrixschenckiisensu stricto in response to oxidative stress induced by hydrogen peroxide

BackgroundSporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. In order to colonize the host, the pathogen must neutralize the reactive oxygen species produced by the phagocytic cells during the respiratory burst. Little is known about these mechanisms in S. schenckii. AimsTo identify the proteins differentially expressed after the exposure of S. schenckiisensu stricto to different concentrations of H2O2. MethodsYeast cells of S. schenckiisensu stricto were exposed to increasing concentrations of H2O2. Proteins differentially expressed in response to oxidative stress were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by MALDI-MS/MS. RT-PCR assays were performed to evaluate the transcription of genes of the identified proteins. ResultsConcentrations of H2O2 as high as 800mM allowed cell growth, and 200mM and 400mM were selected for comparative analysis by 2D-PAGE. This analysis revealed at least five differentially expressed proteins, which were identified as heat shock 70kDa protein (Hsp70), chaperonin GroEL, elongation factor 1-β (EF1-β), a hypothetical protein, and mitochondrial peroxiredoxin (Prx1). RT-PCR revealed that the transcription of the genes coding for some of these proteins are differentially regulated. ConclusionsBased on these results, it is proposed that these proteins may be involved in the resistance of S. schenckii to oxidative stress, and play an important role in the fungus survival in the host.

Sporothrix brasiliensis induces a more severe disease associated with sustained Th17 and regulatory T cells responses than Sporothrix schenckii sensu stricto in mice

Little is known about the differences in the CD4+ T-cell response induced by Sporothrix schenckii and Sporothrix brasiliensis, the most virulent species that cause sporotrichosis. Here, the helper (Th) and regulatory T cells (Tregs) responses were evaluated comparatively in a murine model of sporotrichosis on days 7, 21 and 35 after subcutaneous infection with either S. schenckii or S. brasiliensis conidia. The fungal load was measured at the site of infection, as well as in the liver and spleen. The Th1/Th17/Tregs responses were analyzed in the spleen, while the level of IL-2, IL-4, IL-6, TNF-alpha, IFN-ɣ, IL-17A and IL-10 cytokines were measured at the local site of infection on 24 h postinfections and in sera on the indicated days. S. brasiliensis caused a longer-lasting infection in the skin and chronic systemic dissemination associated to more severe granulomatous lesions. Similar Th1/Th1-Th17/Tregs responses were induced by both S. brasiliensis and S. schenckii on 7th and 21st d.p.i but on 35 d.p.i a reduction of Th1 and Th1-Th17 cells, associated to higher values of Th17/Tregs cells was observed only in S. brasiliensis-infected mice. In summary, S. brasiliensis caused a more severe disease associated with sustained Th17/Tregs responses than S. schenckii in mice.

Dectin-1 expression by macrophages and related antifungal mechanisms in a murine model of Sporothrix schenckii sensu stricto systemic infection.

The available information about the role of Dectin-1 in sporotrichosis is scarce. Hence, we aimed to assess Dectin-1 expression by macrophages and the activation of some related antifungal mechanisms during the Sporothrix schenckii sensu stricto infection as a first attempt to elucidate the role of this receptor in sporotrichosis. Balb/c mice were intraperitoneally infected with S.schenckii sensu stricto yeast ATCC 16345 and euthanized on days 5, 10 and 15 post-infection, when the following parameters were evaluated: fungal burden in spleen, Dectin-1 expression and nitric oxide (NO) production by peritoneal macrophages, as well as IL-1β, TNF-α and IL-10 exvivo secretion by these same cells. Peritoneal macrophages were exvivo challenged with either the alkali-insoluble fraction (F1) extracted from the S.schenckii cell wall, a commercially available purified β-1,3-glucan or whole heat-killed S.schenckii yeasts (HKss). Additionally, a Dectin-1 antibody-mediated blockade assay was performed on day 10 post-infection to assess the participation of this receptor in cytokine secretion. Our results showed that Dectin-1 expression by peritoneal macrophages was augmented on days 10 and 15 post-infection alongside elevated NO production and exvivo secretion of IL-10, TNF-α and IL-1β. The antibody-mediated blockade of Dectin-1 inhibited cytokine production in both infected and non-infected mice, mainly after β-1,3-glucan stimulation. Our results suggest a role for Dectin-1 in triggering the immune response during S.schenckii infection.

Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells.

Sporothrix schenckii sensu stricto and S. brasiliensis are usually associated to sporotrichosis, a subcutaneous mycosis worldwide distributed. Comparative analyses between these two species indicate they contain genetic and physiological differences that are likely to impact the interaction with host cells. Here, we study the composition of the cell wall from conidia, yeast-like cells and germlings of both species and found they contained the same sugar composition. The carbohydrate proportion in the S. schenckii sensu stricto wall was similar across the three cell morphologies, with exception in the chitin content, which was significantly different in the three morphologies. The cell wall from germlings showed lower rhamnose content and higher glucose levels than other cell morphologies. In S. brasiliensis, the wall sugars were constant in the three morphologies, but glucose was lower in yeast-like cells. In S. schenckii sensu stricto cells most of chitin and β1,3-glucan were underneath wall components, but in S. brasiliensis germlings, chitin was exposed at the cell surface, and β1,3-glucan was found in the outer part of the conidia wall. We also compared the ability of these cells to stimulate cytokine production by human peripheral blood mononuclear cells. The three S. schenckii sensu stricto morphologies stimulated increased levels of pro-inflammatory cytokines, when compared to S. brasiliensis cells; while the latter, with exception of conidia, stimulated higher IL-10 levels. Dectin-1 was a key receptor for cytokine production during stimulation with the three morphologies of S. schenckii sensu stricto, but dispensable for cytokine production stimulated by S. brasiliensis germlings. TLR2 and TLR4 were also involved in the sensing of Sporothrix cells, with a major role for the former during cytokine stimulation. Mannose receptor had a minor contribution during cytokine stimulation by S. schenckii sensu stricto yeast-like cells and germlings, but S. schenckii sensu stricto conidia and S. brasiliensis yeast-like cells stimulated pro-inflammatory cytokines via this receptor. In conclusion, S. brasiliensis and S. schenckii sensu stricto, have similar wall composition, which undergoes changes depending on the cell morphology. These differences in the cell wall composition, are likely to influence the contribution of immune receptors during cytokine stimulation by human monocytes.

Molecular Identification and Phenotypic Characterisation of Sporothrix globosa from Clinical Cases of Eastern Assam, North-east India

Sporotrichosis is known to be endemic in the state of Assam, North-east India, which is situated in the Sub-Himalayan region. This disease is an acute or chronic infection caused by Sporothrix schenckii species complex which currently includes several species of clinical relevance such as Sporothrix brasiliensis, Sporothrix globosa, Sporothrix schenckii sensu stricto, Sporothrix albicans, Sporothrix mexicana, Sporothrix pallida and Sporothrix luriei. S. globosa is the prevalent species in India. Eight culture-positive patients were diagnosed from suspected consecutive cases of two lymphocutaneous and six fixed cutaneous forms over a period of 4 years in a clinical mycology laboratory of a tertiary care centre in Eastern Assam. Phenotypic speciation was inconclusive using the criteria of Marimon et al. because of atypical growth pattern shown by the isolates. Our isolates showed good growth at 37°C ranging from 6 to 27 mm; four of the isolates showed growth of 11–27 mm unlike S. globosa strains reported earlier. Molecular identification was done by sequencing both the internal transcribed spacer (ITS) region and the calmodulin (CAL) protein encoding gene (partial). All the isolates were identified as S. globosa. Molecular confirmation of species using ITS region and CAL protein encoding gene (partial) is necessary for isolates of S. globosa showing atypical biopatterns.

A case of lymphocutaneous sporotrichosis caused by Sporothrix schenckii sensu stricto finally diagnosed by molecular identification based on "ITS" and "CAL" coding regions in combination with phenotypic analysis

A 54-year-old female farmer presented with a pea-sized red nodule on the left upper limb near the wrist for 15 days, which occurred after trauma, gradually became swollen and ruptured, and developed into multiple nodules arranged in a chain in 30 days. Skin examination revealed multiple hard purple-red nodules arranged in a line on her left upper limb, some of which were ruptured with a small amount of purulent exudate. Histopathological examination further revealed that the focus of infection manifested as pyogenic granuloma-like inflammation mainly infiltrated with mixed inflammatory cells. Periodic acid Schiff (PAS) staining showed no fungal structures, including fungal spores, hyphae and asteroid body. The biopsy tissue culture yielded the fungus. According to the morphological analysis of the cultures and results of molecular identification based on internal transcribed spacer (ITS) and calmodulin (CAL) coding regions, this case was finally diagnosed as lymphocutaneous sporotrichosis caused by Sporothrix schenckii sensu stricto. The patient was treated with oral potassium iodide 10% solution in a dose of 10 ml thrice a day. After 2-month treatment, the patient felt that the lesions were obviously improved, but afterwards she was lost to follow-up. This research report suggests that phenotypic analysis combined with ITS/CAL-based molecular identification can accurately identify Sporothrix schenckii complex at the species level. Key words: Sporotrichosis; Sporothrix; Phenotype; Sequence alignment; Sporothrix schenckii sensu stricto

The NLRP3 inflammasome contributes to host protection during Sporothrix schenckii infection.

Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen-associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated with fungal recognition is the nucleotide-binding oligomerization domain-like receptor (NLR). After PAMP recognition, NLR family pyrin domain-containing 3 (NLRP3) binds to apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 to form the NLRP3 inflammasome. When activated, this complex promotes the maturation of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and cell death through pyroptosis. In this study, we aimed to evaluate the importance of the NLRP3 inflammasome in the outcome of S. schenckii infection using the following three different knockout (KO) mice: NLRP3-/- , ASC-/- and caspase-1-/- . All KO mice were more susceptible to infection than the wild-type, suggesting that NLRP3-triggered responses contribute to host protection during S. schenckii infection. Furthermore, the NLRP3 inflammasome appeared to be critical for the ex vivo release of IL-1β, IL-18 and IL-17 but not interferon-γ. Additionally, a role for the inflammasome in shaping the adaptive immune response was suggested by the lower frequencies of type 17 helper T (Th17) cells and Th1/Th17 but not Th1 cells in S. schenckii-infected KO mice. Overall, our results indicate that the NLRP3 inflammasome links the innate recognition of S. schenckii to the adaptive immune response, so contributing to protection against this infection.

The Current Status of Feline Sporotrichosis in Malaysia.

Feline sporotrichosis has been reported in Malaysia since the 1990's. Since then, studies have revealed that clinical clade D, Sporothrix schenckii sensu stricto, of a single clonal strain is the most common cause of this disease in Malaysia. The prevalence of a single clonal strain from a clinical clade was never before reported in Asia in a specific geographical niche. This raises the possibility of a process of purifying selection and subsequent clonal proliferation. While agricultural practices may serve as the selective pressure, direct causality has yet to be established. Studies into the thermo-tolerability of the Malaysian clonal strain of S. schenckii sensu stricto revealed that a small minority of clinical isolates have the capacity to grow at 37℃, while the majority displayed low susceptibility to commonly used antifungals in clinical practice, such as itraconazole (ITZ) and terbinafine (TRB). Despite unestablished breakpoints, suspected resistance (MIC > 4 mg/mL) towards amphotericin B (AMB) and fluconazole (FLC) was recorded in the isolates. This explains the often lack of clinical response in feline patients treated with recommended doses of antifungals, including ITZ. Coupled with the potential zoonotic transmission to clients and veterinarians, protracted treatment period, and subsequent cost of treatment, prognosis of feline sporotrichosis is often regarded to be poor. The use of a higher dose of ITZ has been reported, and an adoption of this high-dose treatment regime is reported in this manuscript, with complete cure achieved in cases of recalcitrant and/or unresponsive feline sporotrichosis, which would otherwise be euthanized.

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

Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells.

Δ(24)-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis.

Inhibition of Δ24-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker® Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.

An Outbreak of Lymphocutaneous Sporotrichosis among Mine-Workers in South Africa.

BackgroundThe largest outbreak of sporotrichosis occurred between 1938 and 1947 in the gold mines of Witwatersrand in South Africa. Here, we describe an outbreak of lymphocutaneous sporotrichosis that was investigated in a South African gold mine in 2011.MethodologyEmployees working at a reopened section of the mine were recruited for a descriptive cross-sectional study. Informed consent was sought for interview, clinical examination and medical record review. Specimens were collected from participants with active or partially-healed lymphocutaneous lesions. Environmental samples were collected from underground mine levels. Sporothrix isolates were identified by sequencing of the internal transcribed spacer region of the ribosomal gene and the nuclear calmodulin gene.Principal FindingsOf 87 male miners, 81 (93%) were interviewed and examined, of whom 29 (36%) had skin lesions; specimens were collected from 17 (59%). Sporotrichosis was laboratory-confirmed among 10 patients and seven had clinically-compatible lesions. Of 42 miners with known HIV status, 11 (26%) were HIV-infected. No cases of disseminated disease were detected. Participants with ≤3 years’ mining experience had a four times greater odds of developing sporotrichosis than those who had been employed for >3 years (adjusted OR 4.0, 95% CI 1.2–13.1). Isolates from 8 patients were identified as Sporothrix schenckii sensu stricto by calmodulin gene sequencing while environmental isolates were identified as Sporothrix mexicana.Conclusions/Significance S. schenckii sensu stricto was identified as the causative pathogen. Although genetically distinct species were isolated from clinical and environmental sources, it is likely that the source was contaminated soil and untreated wood underground. No cases occurred following recommendations to close sections of the mine, treat timber and encourage consistent use of personal protective equipment. Sporotrichosis is a potentially re-emerging disease where traditional, rather than heavily mechanised, mining techniques are used. Surveillance should be instituted at sentinel locations.