PCR diagnosis based on the amplification of the fungal gene sequences is a powerful tool for identifying invasive mycoses. The first PCR for identification of S. schenckii sensu lato was described by Kano et al.29 Specific oligonucleotide primers based on the chitin synthase 1 gene were designed, and PCR was able to detect a 10pg genomic DNA fragment of S. schenckii. After, this methodology was employed for the molecular diagnosis of sporotrichosis.28 A nested PCR assay for the detection of S. schenckii was evaluated in clinical samples using the 18S rRNA gene sequence as the target. The nested PCR could detect S. schenckii DNA in tissue samples from infected animals or from clinical specimens from patients with sporotrichosis confirmed by culture or hystochemical staining. The test showed high sensitivity and specificity, indicating that the assay could provide rapid diagnosis with sufficient accuracy to be clinically useful for patients with sporotrichosis.25 More recently, the same assay was used to detect S. schenckii DNA from 38 strains (including all 24 mitochondrial DNA types) collected from different areas of the world, and of tissues of experimentally infected mice, skin biopsies of nine patients with sporotrichosis, two strains of Ceratocystis minor, and 10 different species of other pathogenic fungi.61 The authors demonstrated that nested PCR could correctly identify all S. schenckii strains and clinical samples, corroborating the data obtained by Hu et al.25 that the nested PCR assay is highly sensitive and specific and is a rapid method for diagnosis of sporotrichosis under contamination free conditions.61 Other studies also reported PCR methods applied in the diagnosis of sporotrichosis. Mendoza et al.41 showed an evolution in the previously described nested PCR.25 However, this study demonstrated that this molecular methodology presented lower efficacy than conventional diagnostic methods, such as the gold standard culture. Liu et al.33 reported a PCR in biopsy tissue using the primer S2-R2 that showed positive bands in twenty-five out of the thirty cases (83.3%). These primers were also successfully used for the diagnosis of feline sporotrichosis, with no cross-reactions with normal cat skin cells or other dimorphic fungi.30 These studies did not permit classification at species level of the Sporothrix complex.

In the last few years, the development of DNA-based methods to identify fungal isolates has led to a decrease in the time-consuming step of morphological identification, while maintaining or improving the specificity, accuracy and sensitivity. Until now, few molecular methods have been applied in the detection of S. schenckii DNA from clinical specimens, and in the identification of Sporothrix spp. in culture (Table 1). Diagnosis using PCR is based on the amplification of fungal gene sequences and is a powerful tool for identifying mycoses. One of the pioneering DNA-based methodologies used for the diagnosis of fungal infections was reported by Sandhu et al.55 who developed 21 specific nucleic acid probes targeting the large subunit of the rRNA gene from several fungi, including S. schenckii. The results show a high level of specificity.55 But preview studies56,57 using mitochondrial DNA analysis have reported heterogeneity profiles in S. schenckii strains. In fact, several molecular taxonomic studies using different methodologies, such as restriction fragment length polymorphism (RFLP) from different gene targets, random amplified polymorphic DNA (RAPD), DNA sequencing of internal transcriber spacer (ITS) regions of the ribosomal RNA gene (rRNA), PCR targeting the DNA topoisomerase II gene, amplified fragment length polymorphism (AFLP), and M13 PCR fingerprinting have demonstrated that S. schenckii isolates present different genetic characteristics, suggesting that they do not belong to the same species.3,26,27,32,39,43,44,56,57,60,64

Hence, Marimon et al.37 supported these findings describing through a combination of phenotypic and genetic features four S. schenckii-related species: S. brasiliensis, S. globosa, S. luriei, and S. mexicana. S. globosa has a worldwide distribution,11,35,46 whereas S. mexicana is restricted to Mexico and S. brasiliensis to Brazil.37 However, a case of human sporotrichosis due to S. mexicana was reported in 2011 by Dias et al. in Portugal.13 The molecular tool proposed to differentiate these closely related species was partial calmodulin (CAL) gene sequencing.37 Therefore, an identification key for the S. schenckii complex that includes morphologic analysis of conidia, auxonogram, genotyping via PCR amplification, and sequencing of the CAL gene. Based on this last analysis, Romeo et al.,54 who were studying the molecular phylogeny and epidemiology of Sporothrix species isolated in Italy, demonstrated that 26 environmental strains co-clustered with S. albicans, and two clinical isolates grouped with S. schenckii sensu stricto.

Additionally, phylogenetic analysis based on the rDNA and the β-tubulin regions from S. albicans, S. pallida and S. nivea revealed a significant similarity among them. Therefore, it has been proposed that all the three species were called S. pallida. Moreover, other three environmental Sporothrix species were described using this molecular approach: Sporothrix stylites, Sporothrix humicola and Sporothrix lignivora. The two first species differ from S. schenckii by production of only hyaline conidia and consequent no darkening of colonies with age. S. lignivora has distinctive conidia that do not match in size and shape with other Sporothrix or Ophiostoma species. Isolates classified as S. humicola in this study were referred before as environmental isolates of S. schenckii. In their study, the authors conclude that β-tubulin sequence analysis is strongly recommended in taxonomic studies from Sporothrix species isolated from the environment.12 In fact, β-tubulin analysis, together with ITS sequencing enable the further description of other two environmental Sporothrix species: Sporothrix brunneoviolacea and Sporothrix dimorphospora.36

After the description of Sporothrix complex, one important question is the search for rapid methods for species identification and typing. Oliveira et al.48 reported a PCR fingerprinting using the universal primer T3B to distinguish among species of the Sporothrix complex. The T3B fingerprinting generated clearly distinct banding patterns, allowing the correct identification of all 35 clinical isolates at the species level, which was confirmed by partial CAL gene sequence analyses. Overall, there was a 100% agreement between the species identification using both genotypic methodologies. These profiles were also able to accurately distinguish the species misidentified by phenotypic analysis. The proposed identification technique is simple, reliable, more rapid, less expensive, and requires less technical expertise than sequencing. The computer scanned PCR profiles generated can form the basis of a computer database, which can be used for future identifications of atypical or unidentifiable Sporothrix isolates. This methodology is supposed to be an ideal routine identification system for clinical mycology laboratories, particularly those with limited facilities or technical expertise. There are only a few molecular tools for Sporothrix typing,31,51 and the development of new typing methods are necessary.

The sexual form of all S. schenckii complex species is unknown nowadays; however, there are strong molecular evidences that this fungus undergoes recombination in nature.42 Molecular analyses from 18S region of ribosomal DNA have shown indirect evidence that the sexual form of S. schenckii was Ophiostoma stenoceras.7 On the other hand, morphological and physiological studies show consistent differences between these two species.14,45 These observations lead to consider as different species the O. stenoceras anamorph and S. schenckii. Meanwhile, other molecular studies6,12,24 reinforce that the S. schenckii teleomorph belongs to the genus Ophiostoma, but it is different from O. stenoceras.

Molecular analysis of S. schenckii can also be linked with fungal virulence. A study about the ribosomal conserved region (i.e., ITS) and the nontranscribed spacer (NTS) demonstrated that a S. schenckii sensu strictu strain from disseminated cutaneous sporotrichosis presents a 10bp deletion in the ribosomal NTS region, when compared to isolates controls obtained from patients with fixed cutaneous sporotrichosis.63 Nucleotide polymorphisms are also able to separate environmental and clinical S. schenckii strains. Two single-base transitions in the D1–D2 domain of rDNA differentiate strains from these groups.10

Rapid amplification of cDNA ends technique was recently applied to characterize the gene sequence of a physiology-related protein in S. schenckii. A progesterone adiponectinQ receptor was found to be responsible for an inhibition of, especially, the yeast form of the fungus.18 However, it is necessary a large investigation about this inhibition, since there are several cases of sporotrichosis in woman, pregnant or not,5,9,15 particularly in the endemic area of sporotrichosis where S. brasiliensis predominates.47

S. schenckii is the only clinically relevant dimorphic fungus without an elucidated genome sequence, thus limiting molecular knowledge about the cryptic species of this complex.53 However, some genes have been recently described, such as the α-subunit of the endoplasmic reticulum glucosidase II,52 the α1,2-mannosyltransferase,23 a cytosolic phospholipase A2,58 and a calcium/calmodulin kinase gene.59 These sequences are potential targets for the development of new identification and typing methods to be applied within the Sporothrix complex species.

Nevertheless, nucleotide probes, specific for the Sporothrix species complex, and DNA amplification procedures, such as PCR, allow more rapid and precise diagnosis, which can lead to earlier treatment. The gold standard in diagnosis continues to be using the culture with the correlation of phenotypic characteristics and molecular data.

The authors declare no conflict of interest.