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[Ecological and epidemiological relationships between Sporothrix schenckii, a fungus pathogenic for man and the Ceratocystis genus].

Sporothrix schenckii is a pathogenic fungus for man and animal. Its perfect form is not yet known. Many studies have been made to compare this fungus to ascomycetes belonging to the genus Ceratocystis. This paper summarizes some important data about the ecology and the epidemiology of the fungal complex Sporothrix-Ceratocystis. Previous results obtained by several groups of workers are strongly in favor of a relationship between the two genera. The epidemiological studies carried out in different areas have shown that Sporothrix schenckii and Ceratocystis stenoceras are frequently isolated from the same fragments of plants such as pine or eucalyptus. According to the time and areas, it appears that there is or there is not a relation between the presence of the fungus on the plants or in the soil and the disease. The former case is well illustrated by the epidemic of the Transvaal between 1940 and 1944. Sporothrix schenckii has been found to be present either in endemic areas like Guatemala (around the Ayarza Lake) or in Alsace, or in Corse where no case has been reported for a long time. In France, except for the curious epidemic observed between the years 1903 and 1912, no or very rare cases have been observed although Sporothrix schenckii is abundant in the soil and injuries with contaminated materials are probably extremely frequent. The presence or even the abundance of the fungus in nature is not enough to explain the development of the disease. Some not well known factors are necessary. So as for all disease, the immunological reaction of the host is important and the defects of these mechanisms must be suspected.

Ascomycota

The combined effect of a cutaneo-lymphatic fungus, Sporothrix schenckii and a lymphatic-dwelling nematode, Brugia Malayi.

Domestic cats were infected with third-stage Brugia malayi in such a way that the parasites were restricted to the regional lymphatics of one hind limb. Later, these cats were exposed on the same leg to the yeast phase of Sporothrix schenckii. Edema and fibrosis were more extensive in cats infected with both Brugia and Sporothrix than in cats with either of these organisms alone. Lesions tended to appear earlier, more consistently and progressed more rapidly in cats with dual infections than in Sporothrix controls. No septic lesions were seen in any Brugia controls. Dissemination of the organism was also greater in animals with dual infections than in those infected only with Sporothrix. On the basis of this study, it appears probable that in certain istances fungi, as well as bacteria, may play an important role in transforming a mild subclinical case of lymphatic filariasis into a serious medical problem and that a filarial infection may greatly exacerbate an existing Sporothrix infection.

Animals

[Occurrence of neuraminidase in Sporothrix schenckii and Ceratocystis stenoceras and its role in ecology and pathomechanism of these fungi (author's transl)].

Some strains of Sporothrix schenckii and one strain of Ceratocystis stenoceras were investigated and the occurrence of the enzyme neuraminidase (= sialidase, N-acetylneuraminate glycohydrolase, EC 3.2.1.18) was proved not only by Warren's thiobarbituric acid assay but also by immunoelectrophoresis technique using monospecific antisera against 25 human glycoproteins, lipoproteins, immunoglobulins, fibrinogen and some other proteins (Table 1). There are also fibrinolytic and lipolytic enzyme activities in strains of Sporothrix chenckii corresponding with similar findings of other authors and also in the Ceratocystis stenoceras strain. The temperature optimum of production of neuraminidase seems to be lower than 37 degrees C (Table 2) and all strains failed to produce the enzyme after cultivation at 37 degrees C during some months. The findings of the existence of neuraminidase in the both species confirm also their close relationship. Because the occurrence of N-acylneuraminate was shown only in animals and in some microorganisms but not in plants it can be suggested that also Sporothrix schenckii and Ceratocystis stenoceras grow on animal or neuraminate containing microbial material but not on plants as it is assumed hitherto. Corresponding to this hypothesis the pathogenicity of Sporothrix schenckii and also the facultative pathogenicity of Ceratocystis stenoceras are to see in connexion with the action of neuraminidase and other enzymes metabolizing animal substrates. It can be concluded that neuraminidase is a possible additional pathogenic factor in sporotrichosis below 37 degrees C.

Ascomycota

Ex vivo determination of potentially virulent Sporothrix schenckii,.

Hyphae from 30 isolants of Sporothrix and Ophiostoma species were washed, dried and pyrolyzed at 350 degrees C. Pyrolysis products were separated on a Carbowax column heated 7.5 degrees C/min to and maintained for 50 min at 160 degrees C. Hydrogen flame detector responses were recorded graphically. Fifteen clinical isolants of S. schenckii from geographically separated sources produced qualitatively identical pyrograms. S. foliorum, 8 avirulent S. schenckii and other Sporothrix species isolants from soils, and Sporothrix states of 6 Ophiostoma species yielded pyrograms readily distinguished from each other and from those of virulent S. schenckii. Taxonomic and clinical implications of the pyrograms are mentioned.

Ascomycota

Granulomatous synovitis and osteitis caused by Sporothrix schenckii.

Sporotrichosis must be considered in the differential diagnosis of granulomatous inflammation involving bones and joints. The organisms are difficult to demonstrate in direct smears and in histiologic sections, but they grow readily on routine fungal culture media. The cases of two patients, one with sporothrix arthritis and one with sporothrix arthritis and osteitis, are presented. The latter patient underwent ten surgical procedures over a period of 6 1/2 years and was treated for tuberculous arthritis without a definite diagnosis before fungal cultures were obtained and Sporothrix schenckii isolated.

Diagnostic Errors

Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis.

The largest recorded epidemic of sporotrichosis in the United States occurred in 1988 and involved a total of 84 cases in 15 states. All cases were associated with Wisconsin-grown sphagnum moss. Twenty-one clinical isolates of Sporothrix schenckii and 69 environmental isolates of Sporothrix spp. from the epidemic were characterized and compared. The environmental isolates were recovered from 102 samples of sphagnum moss and other material by using direct plating techniques. Characteristics examined included macroscopic and microscopic morphology, conversion to a yeast phase, exoantigen reactions, and virulence in mice. On the basis of these studies, eight environmental isolates were identified as S. schenckii, five were identified as Ophiostoma stenoceras, and the remainder were identified as Sporothrix species. The environmental isolates of S. schenckii were recovered from moss samples from one Pennsylvania nursery and from three New York State Soil and Water Conservation districts, but none were recovered from moss directly from the bogs in Wisconsin.

Disease Outbreaks

Sporothrix tenosynovitis--differential diagnosis of granulomatous inflammatory disease of the joints.

Sporothrix tenosynovitis is difficult to distinguish from rheumatoid arthritis--the organisms are scarce in biopsy specimens, and both diseases cause a granulomatous synovitis which can affect multiple joints, have associated subcutaneous nodules, similar radiographic findings, and respond to heat and salicylates. This report describes a patient treated for over 2 years as having rheumatoid arthritis. Multiple tenosynovectomy specimens revealed granulomatous synovitis with sterile cultures. Eventually a fungal culture was positive for Sporothrix. This report discusses the histopathologic features of Sporothrix synovitis, and stresses the importance of repeated fungal cultures from patients with granulomatous synovitis unresponsive to steroids.

Antifungal Agents

Molecular and cellular events during the germination of conidia of Sporothrix schenckii.

Hyaline, non pigmented microconidia of Sporothrix schenckii were harvested and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 degrees C. These conditions supported only the development of the mycelial form of Sporothrix schenckii in a reproducible, synchronized manner which allowed further analysis of the early cellular events occurring during the germination of the conidia. The relationship between macro-molecular synthesis (DNA, RNA and protein synthesis) and nuclear division, hyphal growth and septum formation were established. Following inoculation, protein synthesis was observed after 10 minutes followed by RNA synthesis, after 1 h and DNA synthesis after 2 h. The first nuclear division was observed during the 9 to 12 h interval after inoculation. Germ tube formation slightly preceeded nuclear division and was first evidenced 9 h after the induction of germination but was not completed until 12 h after inoculation. Septation was first observed in the germ tubes 0.25 micron from the mother cell-germ tube function 9 h after induction of germination.

Cell Division

Isolation of Sporothrix fungorum from a 500-year-old mummy found in Greenland.

A 500-year-old mummy of a seven-month-old Eskimo infant recently removed from a tomb north of Umanak presented problems of preservation. Among these was fungal coating on the skin, especially on the face. The fungus was isolated and diagnosed as a Sporothrix fungorum. The skin was successfully treated with nystatine. The species Sporothrix fungorum isolated is considered to be very rare, having been reported previously only from Europe and South Africa, but never from Greenland.

Greenland

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.

Antibodies, Fungal

Use of a mouse model to evaluate clinical and environmental isolates of Sporothrix spp. from the largest U.S. epidemic of sporotrichosis.

Five clinical and 69 environmental isolates from the largest U.S. epidemic of sporotrichosis were evaluated in NYLAR male mice following intravenous injection of 5 x 10(6) to 2 x 10(8) conidia per mouse. The clinical isolates and eight environmental isolates produced 100% mortality in groups of three mice each between 12 and 24 days after injection. These virulent isolates grew at 37 degrees C, were dematiaceous by virtue of melanin (melanized) on permissive media (e.g., potato dextrose agar), produced ovoid conidia borne sympodially on lateral conidiophores and pleurogenously about the main hyphal axis, and were identified as Sporothrix schenckii. Two melanized environmental isolates that grew at 35 degrees C but not at 37 degrees C were not virulent and had subtle morphological differences from S. schenckii. The remaining environmental isolates were not melanized, were not virulent, and were not S. schenckii; five were identified as Ophiostoma stenoceras and the remainder were identified as Sporothrix spp. Quantitative organ cultures revealed that clinical isolates grew exponentially in livers and testes, in contrast to an isolate of O. stenoceras that was eliminated from liver, lung, and spleen but that persisted in the testes throughout the 14-day sample period. This model helped to confirm the identification of S. schenckii isolates obtained from the environment.

Animals

Freeze-fracture electron microscopic studies of age-related plasma membrane changes in Sporothrix schenckii.

Characteristics of the plasma membrane of Sporothrix scheckii cells as revealed by freeze-fracture techniques have been classified into eight types (Y1, Y2a, Y2b, Y3a, Y3b, Y4a, Y4b, and Y5) in yeastlike cells grown under the following two conditions: brain heart infusion agar medium at 27 degrees C, and brain heart infusion agar medium at 37 degrees C. Type Y1 cells are yeastlike cells having smooth plasma membranes without any invagination. Typical characteristics of the other types are as follows: type Y2a, smooth plasma membranes with few trenchlike invaginations; type Y2b, wavy plasma membranes with few oval or irregularly formed invaginations; type Y3a, plasma membranes with many randomly distributed trenchlike invaginations; type Y3b, plasma membranes with many cocoonlike or irregularly formed invaginations; type Y4a, plasma membranes with longer trenchlike invaginations; type Y4b, plasma membranes with irregularly formed, enlarged invaginations; and type Y5, smooth or wavy plasma membranes with aggregations of intramembranous particles and with many vacuoles between cell walls and plasma membranes or in the cytoplasm in some cells. By counting the proportion of each type of yeastlike cell under the two conditions and with different cultivation periods, it appears that plasma membrane types change as aging progresses in the following order: type Y1, Y2a, Y3a, Y4a, and Y5 in conidia and type Y1, Y2b, Y3b, Y4b, and Y5 in yeastlike vegetative cells. These observations provide us with an important advantage when studying the effects of antifungal agents on the plasma membrane of Sporothrix scheckii, as it is important to know the natural course of changes in membrane structure during aging.

Cell Membrane

Atigenic similarity between Ceratocystis species and Sporothrix schenckii as observed by immunofluorescence.

Antigenic properties of Sporothrix schenckiii and 2 species of Ceratocystis (C. stenoceras and C. ulmi) were compared by indirect immunofluorescent staining technique. Both species of Ceratocystis and 2 strains of S. schenckii reacted equally well with the unabsorbed antisera against 2 strains of S. schenckii, indicating that these three species share common antigen. The absorption experiments showed that only S. schencki human strain demonstrated a unique antigen. These results support the hypothesis of Ceratocystis-Sporothrix complex in the point that both species have one major common antigen.

Antigens, Fungal

Sporothrix schenckii--a freeze-fracture study.

Freeze-fracture electron microscopy of a pathogenic dimorphic fungus Sporothrix schenckii revealed planar views of cell structures corresponding to those described already on thin sections. In addition to the characteristic differences in cell wall thickness between conidia, yeast forms and filaments, variations in plasma membrane invaginations were found. In conidia the invaginations were short and abundant, while in yeast forms they were scarce and longer. The plasma membrane of the filaments was smooth without invaginations. No differences were found in the frequency of intramembrane particles among the three forms. In the region of the septal pore the particles were circularly arranged with a characteristic partitioning on the P and E fracture faces.

Cell Membrane

Sporothrix schenckii var luriei as the cause of sporotrichosis in Italy.

The second known case of sporotrichosis caused by Sporothrix schenckii var. luriei in a patient living in Piacenza, Italy is described. In the absence of cultures, the diagnosis was based on histologic studies. Stained tissue sections (Hematoxylin and eosin, & Gomori methenamine silver) revealed hyaline, large, thick walled tissue form cells that had divided by septation or a budding process. These forms, along with the striking "eyeglass" configuration of incompletely separated cells that were also present, are the diagnostic features of this apparently rare variety. The use of a fluorescent antibody reagent, specific for S. schenckii, confirmed the identity of the etiologic agent.

Aged

Phylogeny and molecular epidemiology of Sporothrix schenckii in Japan.

Mitochondrial DNA(mtDNA) diversity was investigated in 257 clinical isolants of Sporothrix schenckii obtained from 4 districts in Japan. S. schenckii was classified into 10 types based on Hae III restriction profiles. Phylogeny of types constructed by the method of Fitch and Margoliash [1] on the estimated sequence divergence within mtDNA using the methods of Nei and Li [2], showed that S. schenckii are grouped into 2 clusters, one group consisting of types 1, 2 and 3, and the other group consisting of the other seven types. In addition, types 1, 2, and 5 were correlated with their geographic origin, whereas type 4 was present throughout Japan.

DNA, Fungal

Analysis of restriction profiles of mitochondrial DNA from Sporothrix schenckii and related fungi.

Restriction profiles by HaeIII of mitochondrial DNA were studied for classification and distinction of Sporothrix schenckii (100 strains), S. schenckii var. luriei (1), S. curviconia (1), S. inflata (7), Ceratocystis stenoceras (17) and C. minor (7). These 6 species showed unique restriction profiles which could be discriminated from each other S. schenckii was further separable into 11 types, S. inflata into 4 types, C. stenoceras into 4 types and C. minor into 7 types based on restriction profile heterogeneity.

Ascomycota

Ultrastructure of Sporothrix schenckii treated with iodine-potassium iodide solution.

The ultrastructural changes produced by iodine-potassium iodide solution on yeast cells of Sporothrix schenckii were investigated by transmission electron microscopy in order to clarify the mechanism of oral potassium iodide therapy for sporotrichosis. Yeast cells were dipped with solutions containing various concentrations of iodine. The rate of germination decreased markedly between the range of iodine concentrations from 0.63 microgram/ml to 5.0 micrograms/ml. No significant ultrastructural changes were seen at the concentration of the iodine of 1.25 microgram/ml (80% germination) or less. In the concentration of 2.5 micrograms/ml (50% germination), normal cells and degenerated cells coexisted. When the cells were treated with 5.0 micrograms of iodine per ml (0% germination) or more, their interior structures were completely destroyed. It is assumed that iodine treatment of the organism causes rapid destruction in the whole cell.

Iodine