Molecular identification of the Sporothrix schenckii complex

Sporotrichosis is a subcutaneous fungal infection caused by a thermally dimorphic aerobic fungus, Sporothrix schenckii. It results from traumatic inoculation or contact with animals. Most cases were reported mainly in the tropics and subtropics. The objective of our study is to assess the clinical characteristic of cutaneous sporotrichosis among our patients. We performed a retrospective review of all cases diagnosed with cutaneous sporotrichosis from July 2004 to June 2010. Patients' medical records were retrieved and analyzed according to demography, preceding trauma, sites of lesions, clinical subtypes, treatment, and clinical response. Nineteen cases were diagnosed with cutaneous sporotrichosis with a male/female ratio of 9:10. Thirteen cases (68.4%) were able to recall preceding trauma, and seven of them reported cat scratches or cat bites. Lymphocutaneous sporotrichosis was observed in 13 cases (68.4%) followed by four cases of fixed cutaneous sporotrichosis and two cases of disseminated sporotrichosis. Histologically, 11 cases (57.8%) demonstrated a granulomatous reaction. Sporothrix schenckii was cultured in 12 cases (63.2%). Thirteen cases (68.4%) were successfully treated with oral itraconazole alone for a mean duration of 15.6 weeks. Two cases with disseminated cutaneous sporotrichosis were treated with intravenous amphotericin B. Lymphocutaneous sporotrichosis was the most common subtype of cutaneous sporotrichosis in our series, with cat scratches or bites being the most common preceding trauma. Oral itraconazole was highly effective for the localized subtypes, whereas intravenous amphotericin B was required in disseminated cutaneous sporotrichosis.

Due to the paucity of fungal elements in the tissue, the sensitivity of histopathological tests for the diagnosis of cutaneous sporotrichosis remains low, particularly for low-virulent Sporothrix species. We retrospectively analysed and re-evaluated the role of histopathological examination in the diagnosis of cutaneous sporotrichosis caused by Sporothrix globosa. Retrospective analysis and pathological reassessment were conducted for 69 sporotrichosis cases caused by S. globosa between 2013 and 2024. Statistical analysis using prevalence ratios was conducted to characterise the clinicopathological and epidemiological aspects. Histopathological examination revealed mixed inflammatory cell infiltration and granulomatous changes in all cases. Periodic acid-Schiff (PAS) staining was performed in 29 tissue specimens, with positivity observed in 4 cases (13.8%, 4/29). Following supplemental staining and re-evaluation of the slides, PAS staining revealed positivity in 50.7% (35/69) of cases, demonstrating yeast forms (91.4%, 32/35) with asteroid bodies (n = 2) and rare hyphae (n = 1). Currently, the role of histopathological examination in the diagnosis of sporotrichosis is limited. Our study showed that the frequency of detecting S. globosa based on histopathological examination is considerably high. Nevertheless, achieving high positive rates necessitates the collaborative efforts of experienced mycologists. The predominance of suppurative granulomas or neutrophils is related to the presence of the fungus in tissue sections from human patients.

Sporotrichosis is a fungal infection caused by the Sporothrix species, which have distinct virulence profiles and geographic distributions. We performed a phylogenetic study in strains morphologically identified as Sporothrix schenckii from clinical specimens in Japan, which were preserved at the Medical Mycology Research Center, Chiba University. In addition, we examined the in vitro antifungal susceptibility and growth rate to evaluate their physiological features. Three hundred strains were examined using sequence analysis of the partial calmodulin gene, or polymerase chain reaction（PCR）method using newly designed species-specific primers; 291 strains were Sporothrix globosa and 9 strains were S. schenckii sensu stricto (in narrow sense, s. s.). S. globosa strains were further clustered into two subclades, and S. schenckii s. s. strains were divided into three subclades. In 38 strains of S. globosa for which antifungal profiles were determined, 4 strains (11%) showed high minimal inhibitory concentration (MIC) value for itraconazole. All tested strains of S. schenckii s. s. and S. globosa showed low sensitivity for amphotericin B. These antifungals are used for treatment of sporotrichosis when infection is severe. S. schenckii s. s. grew better than S. globosa; wherein S. globosa showed restricted growth at 35℃ and did not grow at 37℃. Our molecular data showed that S. globosa is the main causal agent of sporotrichosis in Japan. It is important to determine the antifungal profiles of each case, in addition to accurate species-level identification, to strategize the therapy for sporotrichosis.

Definitive diagnosis of sporotrichosis is based on fungal detection in culture. Microscopic methods for the detection of Sporothrix yeast cells in clinical samples have low sensitivity. Although culture methods have high sensitivity, they also have some limitations, such as the time required to conclude the diagnosis, usually from 10 to 15 days, and the difficulty of obtaining an adequate clinical specimen for the test in cases of extracutaneous sporotrichosis. Serological methods are useful tools for a presumptive diagnosis of this infection. The most-used antigenic Sporothrix molecules are the peptide-rhamnomannan and secreted exoantigens. The enzyme-linked immunosorbant assay (ELISA) technique using the peptide-rhamnomannan has high efficiency, and it is useful in the serological follow-up of infection. Exoantigens were first used in immunoprecipitation and agglutination tests, but they have been used more recently in immunoenzymatic tests, with high sensitivity and specificity for both human and feline disease. A glycoprotein of 70 kDa was purified from Sporothrix exoantigens, presenting high immunogenicity, which allows its use in the development of more sensitive and specific methods for sporotrichosis serodiagnosis. Molecular methods of diagnosis can lower the time for diagnosis conclusion, but described methodologies in this field are scarce. In conclusion, the diagnosis of sporotrichosis is a challenging field, and the development of new serological and molecular diagnostic methods is mandatory.

In the 1960s and 1970s, amphotericin B was the only effective therapy for serious systemic endemic fungal infections due to Histoplasma capsulatum, Blastomyces dermatitidis, and Sporothrix schenckii. In the 1980s, ketoconazole was introduced as therapy for endemic mycoses; after this antifungal agent was introduced, some of these infections could be treated orally in an outpatient setting rather than intravenously in an inpatient setting. The 1990s have become the triazole era. It is now standard practice to treat nonmeningeal, non-life-threatening histoplasmosis and blastomycosis orally on an outpatient basis; the drug of choice for this treatment is itraconazole. Itraconazole also has proved useful as treatment for histoplasmosis in patients infected with human immunodeficiency virus. Although itraconazole has not yet been approved for the treatment of sporotrichosis, in preliminary studies it has been shown to be effective therapy not only for cutaneous and lymphocutaneous sporotrichosis but also for disseminated infection with S. schenckii.

Contemporary techniques for molecular diagnoses of mycoses

HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP

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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.

Protein reagents are indispensable for most molecular and synthetic biology procedures. Most conventional protocols rely on highly purified protein reagents that require considerable expertise, time, and infrastructure to produce. In consequence, most proteins are acquired from commercial sources, reagent expense is often high, and accessibility may be hampered by shipping delays, customs barriers, geopolitical constraints, and the need for a constant cold chain. Such limitations to the widespread availability of protein reagents, in turn, limit the expansion and adoption of molecular biology methods in research, education, and technology development and application. Here, we describe protocols for producing a low-resource and locally sustainable reagent delivery system, termed "cellular reagents," in which bacteria engineered to overexpress proteins of interest are dried and can then be used directly as reagent packets in numerous molecular biology reactions, without the need for protein purification or a constant cold chain. As an example of their application, we describe the execution of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) using cellular reagents, detailing how to replace pure protein reagents with optimal amounts of rehydrated cellular reagents. We additionally describe a do-it-yourself fluorescence visualization device for using these cellular reagents in common molecular biology applications. The methods presented in this article can be used for low-cost, on-site production of commonly used molecular biology reagents (including DNA and RNA polymerases, reverse transcriptases, and ligases) with minimal instrumentation and expertise, and without the need for protein purification. Consequently, these methods should generally make molecular biology reagents more affordable and accessible. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cellular reagents Alternate Protocol 1: Preparation of lyophilized cellular reagents Alternate Protocol 2: Evaluation of bacterial culture growth via comparison to McFarland turbidity standards Support Protocol 1: SDS-PAGE for protein expression analysis of cellular reagents Basic Protocol 2: Using Taq DNA polymerase cellular reagents for PCR Basic Protocol 3: Using Br512 DNA polymerase cellular reagents for loop-mediated isothermal amplification (LAMP) Support Protocol 2: Building a fluorescence visualization device.

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are etiological agents of sporotrichosis, a human subcutaneous mycosis. Although the protocols to evaluate Sporothrix virulence in animal models are well described, the cell preparation before inoculation is not standardized, and several culturing media are used to grow yeast-like cells. Here, we found that carbon or nitrogen limitation during fungal cell preparation negatively impacted the ability of S. schenckii and S. brasiliensis to kill Galleria mellonella larvae, but not S. globosa. The fungal growth conditions associated with the short median survival of animals were accompanied by increased hemocyte countings, phenoloxidase activity, and cytotoxicity. The fungal growth under carbon or nitrogen limitation also affected the cell wall composition of both S. schenckii and S. brasiliensis and showed increased exposure of β-1,3-glucan at the cell surface, while those growing conditions had a minimal impact on the S.globosa wall, which had higher levels of this polysaccharide exposed on the wall regardless of the culture condition. This polysaccharide exposure was linked to the increased ability of insect hemocytes to uptake fungal cells, suggesting that this is one of the mechanisms behind the lower virulence of S.globosa or cells from the other species grown in carbon or nitrogen limitation.

Sporotrichosis is an infection caused by the microscopic fungus, Sporothrix schenckii. The disease follows the traumatic inoculation of fungus through injuries involving soil, inhalation of conidia, or zoonotic transmission especially from cat scratches. The objective of the retrospective cohort study was to investigate Th1, Th17, and Treg cell counts and host immunity in patients with lymphocutaneous sporotrichosis. From January 2017 to December 2018, 88 patients, diagnosed with sporotrichosis, were retrospectively reviewed. The patients were divided into acute (≤3 months; n = 46) and non-acute (> 3 months; n = 42) groups based on duration of the disease. We also selected 46 healthy adult participants (control group) for comparison. Th1, Th17, and Treg subsets were tested using flow cytometry (p < 0.05 was considered statistically significant). The Th1 and Th17 cell counts of the acute group were higher than those of the control group (p < 0.05). The Th1 and Th17 cell counts of the non-acute group were lower than those of the control controls (p < 0.05). The longer the duration of disease, the lower the Th1 and Th17 cell counts, however, Treg cell counts were lower in the acute group and higher in the non-acute group, relative to the control group (p < 0.05). An imbalance of Th1, Th17, and Treg cells was found in patients with lymphocutaneous sporotrichosis. The severity and duration of the disease may be affected by the imbalance of Th1, Th17, and Treg cells.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the genus Sporothrix. Since 1998, the number of cases of sporotrichosis due to Sporothrix brasiliensis has grown significantly in Rio de Janeiro, Brazil. Nearly all cases are related to cats as the main source of fungal infection. We report two cases of sporotrichosis following tattoos, a transmission form of S. brasiliensis not yet reported. The first patient, a 22-year-old female, had cutaneous sporotrichosis, fixed form, over a tattoo in her lumbar region. The lesion appeared 12 weeks after she was tattooed. The second patient, a 27-year-old female, had a lymphocutaneous sporotrichosis over a forearm tattoo. The lesion appeared two weeks after she was tattooed. In both cases there was no history of contact with cats or other plausible source of infection. The present study highlights that other non-zoonotic forms of transmission of S. brasiliensis may occur in endemic areas.

Sporotrichosis is an endemic mycosis caused by the species of the Sporothrix genus, and it is considered one of the most frequent subcutaneous mycoses in Mexico. This mycosis has become a relevant fungal infection in the last two decades. Today, much is known of its epidemiology and distribution, and its taxonomy has undergone revisions. New clinical species have been identified and classified through molecular tools, and they now include Sporothrix schenckii sensu stricto, Sporothrix brasiliensis, Sporothrix globosa, and Sporothrix luriei. In this article, we present a systematic review of sporotrichosis in Mexico that analyzes its epidemiology, geographic distribution, and diagnosis. The results show that the most common clinical presentation of sporotrichosis in Mexico is the lymphocutaneous form, with a higher incidence in the 0–15 age range, mainly in males, and for which trauma with plants is the most frequent source of infection. In Mexico, the laboratory diagnosis of sporotrichosis is mainly carried out using conventional methods, but in recent years, several researchers have used molecular methods to identify the Sporothrix species. The treatment of choice depends mainly on the clinical form of the disease, the host’s immunological status, and the species of Sporothrix involved. Despite the significance of this mycosis in Mexico, public information about sporotrichosis is scarce, and it is not considered reportable according to Mexico’s epidemiological national system, the “Sistema Nacional de Vigilancia Epidemiológica.” Due to the lack of data in Mexico regarding the epidemiology of this disease, we present a systematic review of sporotrichosis in Mexico, between 1914 and 2019, that analyzes its epidemiology, geographic distribution, and diagnosis.

Sporotrichosis is a subcutaneous mycosis caused by members of the genus Sporothrix, among which Sporothrix globosa (S. globosa) is the predominant etiological agent in Asia. T cell immunity plays crucial roles in fungal infections, yet the local T cell immune profile in sporotrichosis lesions remains unclear. IL-18, a pleiotropic cytokine capable of modulating T cell responses, is also poorly understood in host defense against S. globosa. qPCR, western blot, and IHC/mIHC were employed to profile IL-18, IL-18 BP, caspase-1 and IL-18R axis, along with Th1,Th2, and Th17 cells and their specific cytokines in sporotrichosis lesions versus healthy skin. Cellular sources of IL-18 in the lesions were identified via mIHC, and IL-18 production from keratinocyte/PBMCs exposed to S. globosa in vitro was tested by qPCR and western blot. Flow cytometry was performed to evaluate the role of IL-18 in Th2 polarization in the PBMCs-S. globosa coculture system. Lesional skin exhibited hyperactivated IL-18 signaling, marked by upregulated IL-18, caspase-1, and IL-18R, alongside decreased IL-18 BP. IL-18 was primarily released by dermal dendritic cells and Langerhans cells, rather than keratinocytes. A mixed Th1/Th2/Th17 microenvironment with Th2 predominance correlated strongly with prolonged disease duration. Strikingly, IL-18 synergistically interacted with Th1-derived IL-2 to establish a self-reinforcing Th2 loop, as evidenced by the strong correlation between lesional IL-4 and IL-18 levels (r = 0.70) and the concomitant upregulation of IL-18/IL-2 during S. globosa-induced Th2 expansion in PBMCs-an effect reversed by IL-18/IL-2 neutralizing antibodies. Critically, this Th2 skewing was mechanistically dependent on NF-κB signaling, as demonstrated through pharmacological pathway inhibition. This study unveils the dual role of IL-18 in human sporotrichosis caused by S. globosa-amplifying both Th1 and Th2 responses but ultimately driving pathogenic Th2 polarization through IL-2 crosstalk. Our work identifies IL-18/IL-2/NF-κB axis as a key Th2-polarizing mechanism driving chronicity in this disease. Targeting this axis could recalibrate anti-fungal immunity, offering translational strategies for this subcutaneous disease.