Fatal pulmonary sporotrichosis caused by Sporothrix schenckii var. luriei in India.

The first case of fatal pulmonary sporotrichosis caused by Sporothrix schenckii var. luriei in a patient from the northwestern region of India is described. In the absence of cultures, the diagnosis was suspected by notation, in lung tissue, of large, thick-walled, hyaline fungal cells that divided internally by septation or a budding process. The thick-walled, internally septated cells often became muriform. The presence of an "eyeglass" configuration of incompletely separated cells characteristic of S. schenckii var. luriei in large numbers aided the diagnosis. The identity of the etiologic agent was confirmed by application of a fluorescent-antibody reagent specific for S. schenckii.     

Sporothrix schenckii Lymphadentitis in a Male with X-linked Chronic Granulomatous Disease

Sporothrix schenckii lymphadenitis was identified in a 33 month old male with X-linked chronic granulomatous disease (CGD). S. schenckii is a dimorphic catalase producing fungus found in the soil of temperate and tropical climates. Host defense against S. schenckii relies primarily on innate and cellular responses and gp91^phox?/? mice are susceptible to disseminated infection. This case represents the first report of susceptibility to sporotrichosis in a patient with CGD.     

Sporothrix schenckii and Sporotrichosis

Sporotrichosis, which is caused by the dimorphic fungus Sporothrix schenckii, is currently distributed throughout the world, especially in tropical and subtropical zones. Infection generally occurs by traumatic inoculation of soil, plants, and organic matter contaminated with the fungus. Certain leisure and occupational activities, such as floriculture, agriculture, mining, and wood exploitation, are traditionally associated with the mycosis. Zoonotic transmission has been described in isolated cases or in small outbreaks. Since the end of the 1990s there has been an epidemic of sporotrichosis associated with transmission by cats in Rio de Janeiro, Brazil. More than 2,000 human cases and 3,000 animal cases have been reported. In humans, the lesions are usually restricted to the skin, subcutaneous cellular tissue, and adjacent lymphatic vessels. In cats, the disease can evolve with severe clinical manifestations and frequent systemic involvement. The gold standard for sporotrichosis diagnosis is culture. However, serological, histopathological, and molecular approaches have been recently adopted as auxiliary tools for the diagnosis of this mycotic infection. The first-choice treatment for both humans and cats is itraconazole.     

Fungemia caused by an amphotericin B-resistant isolate of Sporothrix schenckii. Successful treatment with itraconazole

A 58-year-old, alcoholic, diabetic man presented with multiple, ulcerated skin lesions and polymicrobial septicemia. Sporothrix schenckii was recovered from blood cultures and was resistant to amphotericin B by in vitro testing. Amphotericin B therapy failed, but the patient responded dramatically to itraconazole therapy, only to relapse 3 months after therapy was stopped. Reinstitution of itraconazole therapy has produced another dramatic response. This report is noteworthy for three reasons. First, to our knowledge, it represents only the second reported instance of fungemia with S schenckii that responded to medical therapy. Second, it illustrates that in vitro antifungal susceptibility tests may predict clinical infection response to drug therapy. Third, it suggests that itraconazole has significant promise in treating systemic sporotrichosis.     

Sporothrix schenckii fungemia without disseminated sporotrichosis.

Fungemia is a rare complication of Sporothrix schenckii infection and has always been associated with disseminated sporotrichosis. We describe an immunocompetent patient with localized lymphocutaneous sporotrichosis from whose blood the fungus was isolated. A lysis-centrifugation blood culture system may have improved our ability to detect low-level S. schenckii fungemia.     

Molecular phylogeny of Sporothrix schenckii

The pathogenic dimorphic fungus Sporothrix schenckii is the agent responsible for sporotrichosis, an important fungal infection with a worldwide distribution. Little is known about the population structure of S. schenckii, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to determine, by sequence analysis of three protein coding loci (chitin synthase, beta-tubulin, and calmodulin), whether this variability is due to species divergence or intraspecific diversity in S. schenckii. We included in the analysis 60 isolates (59 of clinical and 1 of environmental origin) of this species from a wide geographical range. DNA sequence data from the three nuclear regions were used in a phylogenetic analysis. The combined analysis of the three loci revealed the presence of three major clades, one grouping all of the European isolates, another with only Brazilian isolates, and the third with isolates from other South American countries and Africa. A total of 14 100% bootstrap-supported nodes were shown, 6 of them representing putative phylogenetic species. Our data also demonstrated that most of these species prevail in different geographical regions.     

Ingestion of yeast forms of Sporothrix schenckii by mouse peritoneal macrophages.

The ingestion by thioglycolate-elicited mouse peritoneal macrophages of yeast forms of two strains of Sporothrix schenckii was studied. Yeast forms opsonized with concanavalin A (ConA) were extensively phagocytized, and the phagocytic indexes depended on the concentration of ConA and apparently on the number of lectin receptors at the yeast surface as well. Neuraminidase treatment of S. schenckii increased the ingestion of unopsonized yeasts 7.7-fold. The addition of monosaccharides and derivatives partially inhibited phagocytosis. Mannose, rhamnose, and galactose, which are major constituents of S. schenckii surface antigens, reduced the phagocytic indexes by 40 to 50%. Glucosamine, N-acetylglucosamine, and N-acetylneuraminic acid were equally effective as inhibitors of phagocytosis. A mixture of five neutral sugars and glucosamine inhibited phagocytosis by 73%. The inhibitory effect of simple sugars could be amplified by using neuraminidase-treated yeast cells. Pentoses and glucose were inactive or slightly inhibitory. A purified rhamnomannan inhibited phagocytosis of the homologous strain, whereas partially purified peptidopolysaccharides were toxic to peritoneal macrophages. A partially purified galactomannan from S. schenckii was inhibitory (62% inhibition), and a peptidopolysaccharide fraction in which the O-linked carbohydrate chains had been removed neither was toxic to macrophages nor inhibited phagocytosis. Pretreatment of macrophages with simple sugars under conditions inhibiting ingestion or binding of S. schenckii did not affect phagocytosis of latex particles or sensitized sheep erythrocytes. The presence of receptors at the peritoneal macrophages which bind S. schenckii cell surface components is suggested.     

Activation of the alternative complement pathway by Sporothrix schenckii

We studied the activation of complement by Sporothrix schenckii yeast cells. Total complement activity, and the effect of various activators on this activity, were assayed on aliquots of fresh nonimmune human serum with and without prior treatment with chelators. Both total hemolytic complement and C3 were consumed (activated) in serum chelated with magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, which blocks the classical pathway but leaves the alternative pathway intact. Further, C3 was consumed, but C4 (exclusively a component of the classical pathway) was preserved, in nonchelated serum after challenge by S. schenckii yeast cells. Absorption of serum with S. schenckii yeast cells to deplete antibodies did not alter these results. Furthermore, immunofluorescence studies demonstrated that C3, but not immunoglobulin G, was deposited on yeast cells during incubation with nonimmune serum. These data indicate that S. schenckii yeast cells activate the alternative complement pathway in vitro independently of antibody. These data do not define a role for the alternative pathway in in vivo host defenses against infection with this organism but provide a foundation for studies to evaluate such a role.     

Molecular epidemiology of Sporothrix schenckii]

Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) of clinical and environmental isolates of Sporothrix schenckii was investigated. Among mtDNA RFLP patterns with Hae III, of 14 environmental isolates morphologically identified as S. schenckii, only 2 isolates were confirmed as S. schenckii, while of more than 500 clinical isolates, all were confirmed to have this condition. Therefore, RFLP analysis of mtDNA is essential for the identification of environmental, but not clinical, isolates of S. schenckii. Isolates of Sporothrix schenckii were classified into 23 mtDNA types (Types l-23) based on mtDNA RFLP patterns with HaeIII and clustered into two major groups by phylogeny, Group A (Types 1-3, 11, 14-19, 22 and 23) and Group B (Types 4-10, 12, 13, 20 and 21). Group A isolates are predominant in South Africa, North America, Central America and South America, while Group B isolates are predominant in Australia and Japan. In Japan, the relative distribution of the mtDNA types varied with geographic region: Types 4, 6 are comparatively abundant in West Japan (Kansai and Kyushu districts), Type 5 is comparatively abundant in East Japan (Tokai, Kanto and Tohoku districts) and Type 2 is abundant in the Hokuriku district. Type 1 is found only in the Hokuriku district.     

Molecular analysis of DNA polymorphism of Sporothrix schenckii.

Partial cDNA cloning of a putative membrane transporter protein gene expressed in Sporothrix schenckii and DNA polymorphism of the isolated gene are described here. DNA fragments were isolated from S. schenckii, and the deduced amino acid sequence from one of the fragments contained a region homologous to the conserved sequence of the membrane transporter protein family. 188-bp fragments encoding the homologous region were amplified from many strains of S. schenckii, and were subjected to polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) analysis. The results demonstrated that the strains of S. schenckii were divided into three groups, and several base substitutions among these groups were observed. This finding agreed with the classification of S. schenckii strains based on the mitochondrial DNA diversity, because the three groups were clearly located on the branches of the phylogenetic tree constructed by digestion profiles of mitochondrial DNA with restriction enzymes. The correlation of the results of PCR-SSCP analysis with the mitochondrial DNA diversity might indicate linkage of the mutation of the membrane transporter protein gene with the evolution of S. schenckii, suggesting the protein plays an essential role in S. schenckii.     

Mitochondrial DNA analysis of Sporothrix schenckii]

Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) of clinical and environmental isolates of Sporothrix schenckii was investigated. Isolates of S. schenckii were classified into 24 mtDNA types (Types 1-24) based on mtDNA RFLP patterns with HaeIII and clustered into two major groups by phylogeny, Group A and Group B. Group A isolates are predominant in South Africa, North America, Central America and South America, while Group B isolates are predominant in Australia, Japan and China. Based on the mtDNA-RFLP patterns with HaeIII, most environmental isolates morphologically identified as S. schenckii were confirmed to be species distinct from S. schenckii and S. schenckii isolates were few, while all of more than 500 clinical isolates were confirmed as S. schenckii. Therefore, RFLP analysis of mtDNA is essential for the identification of environmental, but not clinical isolates of S. schenckii.     

Long-chain fatty acids of Sporothrix (Sporotrichum) schenckii.

A number of strains purporting to belong to the species Sporothrix schenckii were examined for their fatty acid content. The majority of the strains were isolated from cases of sporotrichosis. Two strains were reputedly saprophytic. In all cases except the two saprophytic ones the major fatty acid was a C18 diene. Considerable amounts of palmitic acid and C18 monoene were found in all strains.     

Sporothrix schenckii sensitivity to voriconazole, itraconazole and amphotericin B.

One hundred clinical isolates of Sporothrix schenckii were tested against voriconazole, itraconazole and amphotericin B using a modification of the NCCLS M27-A in vitro yeast susceptibility testing procedure. NCCLS M38-P for moulds was not used because yeast forms may have been present when the test isolates were incubated at 35 +/- 1 degrees C. The minimum inhibitory concentration (MIC) values were: voriconazole 0.5-8 (geometric mean titer 6.50) microg ml(-1) ; itraconazole 0.03-8 (geometric mean titer 1.56) microg ml(-1); and amphotericin B 0.25-2 (geometric mean titer 1.23) microg ml(-1). The minimum fungicidal concentration (MFC) values were: voriconazole 2-8 (geometric mean titer 7.67) microg ml(-1); itraconazole 0.125-8 (geometric mean titer 7.41) microg ml(-1); and amphotericin B 0.125-2 (geometric mean titer 1.53) microg ml(-1). Based upon MIC values, sensitivity to amphotericin B is strain-dependent. S. schenckii is more sensitive to itraconazole than voriconazole based upon a comparison of MIC geometric means, even though the MIC ranges were essentially the same.     

Biosynthesis and functions of melanin in Sporothrix schenckii

Sporothrix schenckii is a human pathogen that causes sporotrichosis, an important cutaneous mycosis with a worldwide distribution. It produces dark-brown conidia, which infect the host. We found that S. schenckii synthesizes melanin via the 1,8-dihydroxynaphthalene pentaketide pathway. Melanin biosynthesis in the wild type was inhibited by tricyclazole, and colonies of the fungus were reddish brown instead of black on tricyclazole-amended medium. Two melanin-deficient mutant strains were analyzed in this study: an albino that produced normal-appearing melanin on scytalone-amended medium and a reddish brown mutant that accumulated and extruded melanin metabolites into its medium. Scytalone and flaviolin obtained from cultures of the reddish brown mutant were identified by thin-layer chromatography, high-performance liquid chromatography, and UV spectra. Transmission electron microscopy showed an electron-dense granular material believed to be melanin in wild-type conidial cell walls, and this was absent in conidial walls of the albino mutant unless the albino was grown on a scytalone-amended medium. Melanized cells of wild-type S. schenckii and the albino grown on scytalone-amended medium were less susceptible to killing by chemically generated oxygen- and nitrogen-derived radicals and by UV light than were conidia of the mutant strains. Melanized conidia of the wild type and the scytalone-treated albino were also more resistant to phagocytosis and killing by human monocytes and murine macrophages than were unmelanized conidia of the two mutants. These results demonstrate that melanin protects S. schenckii against certain oxidative antimicrobial compounds and against attack by macrophages.     

Environmental Isolates of Sporothrix schenckii in China]

Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) from 25 environmental isolates of Sporothrix schenckii from northeastern China was investigated. Based on the mtDNA-RELP patterns with Hae III, 6 isolates were confirmed to be S. schenckii, while the other 19 isolates were confirmed to be species distinct from S. schenckii. The mtDNA RFLP patterns of the 19 non-S. schenckii were identical to each other. The non-S. schenckii isolates could not be discriminated from S. schenckii by their macro- or micro-morphological features, and were not pathogenic in guinea pigs. Serological and delayed hypersensitivity cross-reactions were found between S. schenckii and the non-S. schenckii species, suggesting antigenic similarity. These results indicate that RFLP analysis of mtDNA is essential for the identification of environmental isolates of S. schenckii.     

Immunoblot analysis of antibody responses to Sporothrix schenckii

The serologic response to Sporothrix schenckii was investigated in patients with sporotrichosis by solid-phase enzyme-linked immunosorbent assays (ELISAs) and Western immunoblot techniques. A soluble antigen preparation derived from an S. schenckii isolate contained 15 protein staining components ranging in molecular size from 22 to 70 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera from 40 patients with sporotrichosis demonstrated Sporothrix immunoglobulin G antibody by ELISA with titers between 128 and 65,200. No sera from 300 healthy individuals or 100 patients with various systemic mycoses other than sporotrichosis had ELISA titers greater than 64. By Western immunoblotting of the antigens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sera from 10 patients with cutaneous sporotrichosis reacted with 8 to 10 antigen components (range, 40 to 70 kDa), while sera from 15 patients with extracutaneous sporotrichosis reacted with a greater number of antigen components (15 to 20 bands) over a wider range of molecular sizes (22 to 70 kDa). Antibody to 40- and 70-kDa antigen components was detected by immunoblots in all sera tested from patients with sporotrichosis. Antibody to 22- to 36-kDa antigen components was present in sera from 13 of 15 patients with extracutaneous sporotrichosis, but these lower-molecular-weight components were not detected by sera from patients with cutaneous sporotrichosis. Antibody to these components was not detected by Western blotting in sera from 19 of 20 patients with other fungal diseases or from 30 healthy individuals. Purification of these specific antigen fractions could provide the basis of a sensitive and specific serodiagnostic test to indicate the presence and activity of extracutaneous sporotrichosis.     

Virulence differences between albino and pigmented Sporothrix schenckii isolates

ObjectiveThis work compares the virulence between pigmented (P) and albino (A) filamentous forms of S. schenckii through experimental skin sporotrichosis.Material and methodsTwo original wild pigmented isolates (1 and 2) cultivated in yeast extract peptone dextrose (YEPD), were exposed to 300 erg/mm² of UV radiation in order to produce mutant albinos. Swiss albino mice were divided in four groups (P1, A1, P2 and A2) and inoculated in the right paw pad with 0.1 ml of solution (1 × 10⁶cells/ml) and observed during nine weeks.ResultsThe albino groups A1 (86%) and A2 (100%) presented intense inflammatory reactions in the inoculation point (p < 0.01) in Week 2 after inoculation, while in the pigmented groups, around 70% of the animals presented multiple skin lesions in Week 4 (p < 0.01). In the last three weeks of the experiment, 67% of the mice in group P1 demonstrated multiple skin lesions. S. schenckii was recovered from samples obtained in necropsy and the histological analysis revealed formation of granulomas with yeast-like cells in the central area of the lesion.ConclusionThe results obtained in this experimental model permit the conclusion that there are virulence differences between pigmented and mutant albino isolates of S. schenckii in the development of experimental skin sporotrichosis.     

Mitochondrial DNA analysis of Sporothrix schenckii in South Africa and Australia.

Clinical isolates of Sporothrix schenckii were investigated for mitochondrial DNA (mtDNA) types using restriction fragment length polymorphism patterns with HaeIII. The 62isolates in South Africa comprised Types 3 (9.7%), 4 (1.6%), 11 (9.7%), 17 (77.4%) and 23 (1.6%) while the 23 Australian isolates comprised Types 3 (26.0%), 4 (56.5%), 7 (8.6%) and 21 (8.6%). In a phylogenetic tree based on the sequence divergence of mtDNA, the mtDNA types were clustered into two groups, A and B. The results suggested that isolates in South Africa mainly belong to Group A and isolates in Australia mainly belong to Group B.     

Analysis of restriction profiles of mitochondrial DNA from Sporothrix schenckii.

Mitochondrial DNA (mtDNA) diversity was analyzed in 42 clinical isolates of Sporothrix schenckii from Mexico (n = 29), Guatemala (n = 4) and Colombia (n = 9). Based on HaeIII restriction digestion profiles, the isolates were classified into eight types. In addition to 24 mtDNA types previously reported in another study, 6 new types were found in this study. Most of the strains belong to type 14 and type 30, the former restricted to Mexico, whereas the latter was distributed in Mexico, Guatemala and Colombia. The new types (25-30) were identified in Mexico, Guatemala and Colombia. Restriction-fragment length polymorphism in mtDNA of S. schenckii revealed high levels of genetic variation attributable to differences in restriction sites as well as in mtDNA size. Based on genetic distances S. schenckii types were clustered into two main groups.