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This article describes PCR fingerprinting using the universal primer T3B to distinguish among species of the Sporothrix complex, S. brasiliensis, S. globosa, S. mexicana, and S. schenckii. This methodology generated distinct banding patterns, allowing the correct identification of all 35 clinical isolates at the species level, confirmed by partial calmodulin (CAL) gene sequence analyses. This methodology is simple, reliable, rapid, and cheap, making it an ideal routine identification system for clinical mycology laboratories.

We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.

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.

Sporothrix schenckii causes sporotrichosis, a disease that most commonly presents as a subacute or chronic skin infection. An unusually high incidence of clinical cases of sporotrichosis occurred in the southwest of Western Australia over the last 5 years. Anecdotal accounts from patients implicated contact with hay prior to infection. Isolates of S. schenckii from hay and clinical cases were investigated by traditional phenotypic methods and pulsed-field gel electrophoresis (PFGE). The phenotypic evaluation separated S. schenckii from Ophiostoma spp. A DNA macrorestriction method using SfiI and NotI macrorestriction digestion by PFGE was developed to investigate the epidemiological connections. BioNumerics software was used to analyze the results. DNA macrorestriction digestion patterns for the recent Western Australian clinical isolates and four hay isolates were indistinguishable. Eastern state clinical isolates, national Quality Assurance Program isolates, and other environmental isolates gave different macrorestriction patterns. Clinical isolates from the southwest of Western Australia collected in the 1980s and 1990s were also characterized using PFGE. The patterns generated were indistinguishable from those of the recent clinical isolates. PFGE showed that the dominant strain of S. schenckii causing sporotrichosis in Western Australia is present in hay, has caused sporotrichosis for at least 15 years, and is a different strain from the strains found in other parts of Australia.

Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of Peruvian strains of Sporothrix schenckii and to compare them to a panel of non-Peruvian strains. AFLP analysis suggests that the Peruvian strains can be divided into two homogeneous clusters with no reference to geographical origin or the clinical form of sporotrichosis. The strains from abroad present heterogeneous profiles, with the Bolivian strain and the Colombian strains related to one of the Peruvian population. Sequencing of internal transcribed spacer 2, used to examine the relationships over a longer distance, confirmed the division of Peruvian strains into two populations that can be identified on the basis of a single but specific sequence divergence. This paper introduces automated AFLP analysis as a valuable tool for further investigation of the epidemiology and ecology of S. schenckii.

Cutaneous sporotrichosis is a chronic granulomatous fungal infection caused by Sporothrix schenckii with worldwide distribution. Its traditional diagnosis is time-consuming and difficult to differentiate from that of a clinical sporotrichoid lesion caused by various pathogens. In this study, a nested PCR assay for the detection of S. schenckii was evaluated by using a sequence of 18S rRNA gene as a target. For the examination of specificity and sensitivity, five clinical isolates with 1 ATCC 10213 strain of S. schenckii, 10 strains of clinical common fungi, 3 strains of Mycobacterium spp., Staphylococcus aureus, and normal human skin tissue were used. The expected fragment was amplified from six S. schenckii isolates in the first round and nested PCR but not from other microorganisms and human DNA. Their sequences were 100% identical to the S. schenckii 18S rRNA gene sequence deposited in GenBank. A detection limit of 40 fg of S. schenckii DNA extract was determined with ethidium bromide staining. Serial dilution studies demonstrated that the nested PCR could detect a DNA amount of 1 CFU of S. schenckii in tissue samples. We further investigated the nested PCR assay for the detection of S. schenckii from the tail tissues of 5 experimentally infected mice and from the clinical biopsy specimens of 12 patients with sporotrichosis confirmed by culture or histochemical staining. The nested PCR assay was positive in all 5 infected mice and in 11 of the 12 clinical specimens. The high sensitivity and specificity of this nested PCR indicate that the assay can provide rapid diagnosis with sufficient accuracy to be clinically useful for patients with sporotrichosis.

Sporothrix schenckii isolates of fixed and lymphocutaneous clinical forms from Mexico (MX), Guatemala (GT), and Colombia (CO) as well as environmental isolates from MX were studied by analyzing their phenotypic characteristics (conidial length, thermotolerance by percent growth inhibition [GI] at 35 and 37 degrees C, median lethal dose [LD(50)]) and genotypic characteristics (by random amplified polymorphic DNA [RAPD] analysis-PCR). A significant difference (P < 0.01) in the mean conidial length of S. schenckii clinical isolates from CO ( = 4.03 +/- 1.04 microm) compared with those of clinical isolates from MX ( = 2.06 +/- 0.53 microm) and GT ( = 2.68 +/- 0.83 microm) was observed. The lowest thermotolerance, as determined by measurement of percent GI, was exhibited by isolates from CO at 35 degrees C ( = 50.1% +/- 15.9%) and 37 degrees C ( = 72.7% +/- 10.9%). In general, the highest virulence, as determined by measurement of the LD(50) for mice, was observed for the MX environmental isolates. RAPD analysis-PCR with 10-mer primers OPBG-01, OPBG-14, and OPBG-19 generated 52 reproducible bands. The 44 Sporothrix isolates fell into four major groups by hierarchical cluster analysis. The first group (group I), formed by 25 (of 27) isolates from MX, had two subgroups: subgroup Ia with 10 environmental isolates and subgroup Ib with 14 clinical isolates. The second group (group II) had two subgroups: subgroup IIa, formed by isolates from CO, and subgroup IIb, formed by isolates from GT. Groups III and IV each had only one clinical isolate from MX. A principal-component analysis of the same data yielded three distinct groups, depending on the geographical origins of the isolates, including the isolates in groups III and IV from MX, which were grouped with the isolates from MX by principal-component analysis. This study revealed that isolates from CO had low thermotolerances at 35 and 37 degrees C and could be associated with superficial skin lesions in patients with fixed clinical forms of sporotrichosis, the most frequent form of the disease in CO. Distinct patterns dependent on geographical origins were also revealed by RAPD analysis-PCR, but these had no relation to the clinical form of the disease.

DNA typing techniques were used to examine selected clinical and environmental isolates of Sporothorix spp. recovered from the 1988 sporotrichosis epidemic in multiple states of the United States. Previous studies indicated that isolates in one of the six morphologically or physiologically distinct groups (group I) obtained from environmental sources were Sporothrix schenckii and were the possible etiologic agents responsible for the epidemic. To assess this hypothesis at the genetic level, whole-cell DNA was extracted from selected clinical isolates and representative members of each of the six environmental groups subjected to endonuclease digestion and then analyzed by gel electrophresis. DNA types were assigned on the basis of restriction fragment length polymorphism patterns. One DNA type was common to clinical and group I isolates but was dissimilar from the DNA types exhibited by groups II to VI. In contrast, a variety of DNA types were associated with isolates in groups II to VI. The latter groups appeared to make up a heterogeneous collection of fungi, with some members of the same group having different DNA types but with others from different groups possessing identical DNA types. Thus, DNA typing studies confirmed that group I environmental isolates are indistinguishable from clinical isolates and that group II to VI isolates represent a complex of related fungi with similar Sporothrix anamorphs.