Keywords

8.1 Introduction

Mycoses can be challenging to diagnose, and accurate interpretation of laboratory data is important to ensure appropriate treatment. Although the clinical manifestations of sporotrichosis are well described, the diagnosis of this mycosis cannot be based on clinical information alone because the symptoms of sporotrichosis overlap with those of other diseases.

Sporotrichosis is classically diagnosed by correlating clinical, epidemiological, and laboratory data (Zancope-Oliveira et al. 2011). Typical laboratory analyses include microscopic examination using 10 % potassium hydroxide or 4 % sodium hydroxide to detect parasitic cigar-shaped, budding, yeast-like cells. These fungal cells are small (2–6 μm in diameter), rare, and difficult to detect during direct examination of specimens obtained from human patients (Kwon-Chung and Bennett 1992) or from domestic animals, such as dogs (Schubach et al. 2006). On the other hand, when this test is performed on skin biopsies collected from cats infected with Sporothrix schenckii, yeast cells are easily observed because cats have a high fungal burden on their lesions (Schubach et al. 2004). Microscopic methods for yeast detection in clinical samples are of low sensitivity (Barros et al. 2011), and asteroid body observation varies among different works (Quintella et al. 2011). However, definitive diagnosis of sporotrichosis is based on fungal detection in culture (Zhang et al. 2011). Cultivation of clinical specimens in mycological media such as Sabouraud dextrose agar or mycobiotic agar yields white filamentous colonies that become brown to black after a few days. Subculturing these colonies in brain–heart infusion at 35–37 °C results in white to creamy yeast-like colonies (Barros et al. 2011). S. schenckii identification is based on the macro- and micromorphologies of the mycelial (Fig. 8.1) and yeast forms (Zancope-Oliveira et al. 2011). However, these characteristics do not differentiate the newly described species of the Sporothrix complex. To physiologically differentiate the species within this complex, other tests such as carbohydrate assimilation (especially sucrose and raffinose), growth rates at 30 and 37 °C, as well as production of dematiaceous sessile conidia are necessary (Marimon et al. 2007, 2008). However, discrepancies between physiological and molecular methods of identification have been described (Oliveira et al. 2011). Moreover, although positive cultures provide the strongest evidence for sporotrichosis, there are some significant limitations. In particular, in some manifestations of the disease, such as S. schenckii–induced arthritis, the collection of material for culture is difficult (Morris-Jones 2002). In addition, sporotrichosis may be mistaken for other infections, such as tuberculosis, leishmaniasis, paracoccidioidomycosis, gummatous syphilis, and chromoblastomycosis (Rippon 1988; Sharma et al. 2005).

Fig. 8.1
figure 1

Morphologic characteristics of the mycelial form of the Sporothrix complex. (a) Sporothrix schenckii sensu stricto colony on potato dextrose agar, incubated at 30 °C for 21 days. (b) Colony of a S. brasiliensis strain, showing a similar morphology to the S. schenckii strain. (c) Slide culture of the S. schenckii strain, showing hyaline conidia on sympodial conidiophores and dematiaceous conidia on denticles arising from the hyphae. Bar 10 μm

Full size image

Non-culture methods have been developed to improve the rate and speed of diagnosis. Additional diagnostic tools are currently available for diagnosis of sporotrichosis to supplement culture and microscopic examination. These laboratory tests have a rapid turnaround time and reasonable specificity and sensitivity. For instance, serological techniques involving antibody detection have been developed using different methodologies. Molecular methods to detect Sporothrix species complex DNA in clinical specimens, including tissue fragments, are also being studied in several laboratories to facilitate rapid diagnosis of infection (Ruiz-Baca et al. 2013; Oliveira et al. 2014). The results from the described tests can provide a presumptive diagnosis of sporotrichosis and require clinical correlation for the correct evaluation and determination of the final diagnosis.

Serological techniques are usually simpler than culture and very useful in the diagnosis and follow-up of patients with sporotrichosis. The following serologic tests have been applied in the diagnosis of Diagnostic methods: agar gel immunodiffusion (ID), slide (SLA), tube (TA) and latex agglutination (LA), complement fixation reaction (CF), immunofluorescence (IF) (Blumer et al. 1973), immunoelectrophoresis (IEP) (Albornoz et al 1984), and immunoenzyme assays in several formats, such as ELISAs, and immunoblots. These techniques have advanced considerably in recent decades because of the development of innovative detection schemes in the identification of relevant Sporothrix spp. antigens. However, the literature concerning the serodiagnosis of sporotrichosis is neither extensive nor diverse.

A range of antigenic preparations, derived both from whole yeast cells and from culture filtrate in their crude and/or purified states, have been applied in serological tests, which has resulted in high cross-reactivity, one of the most persistent problems found in the serodiagnosis of sporotrichosis. Moreover, these antigenic preparations are highly variable, making it very difficult to standardize diagnostic techniques in different laboratories.

The next sections focus on the main antigens and serologic methods applied for the presumptive diagnosis of sporotrichosis, as well as the application of some antigenic preparations in skin tests and molecular diagnosis.

8.2 Antigen Detection

The antigenic composition of the members of the Sporothrix complex is poorly understood. The most studied molecules with immunological reactivity of these fungi are the peptide-rhamnomannan, the exoantigens, and the newly described gp-70.

The Sporothrix peptide-rhamnomannan is a fraction of the fungal cell wall that presents an affinity to concanavalin A(Con-A) and that reacts with antibodies present in sera from patients with sporotrichosis (Lloyd and Bitoon 1971). Through a western blot analysis, this Con-A binding fraction of the Sporothrix cell wall was characterized as a mixture of three antigenic fractions of 84, 70, and 58 kDa. Moreover, through a β-elimination procedure, it was verified that the 70-kDa molecule is a deglycosylated form of the 84-kDa antigen (Lopes-Bezerra and Lima 1997). This antigen is stable, and its immunological reactivity does not significantly change if different Sporothrix strains are used in the extract preparation (Bernardes-Engemann et al. 2009). The potential application of this antigen to the serodiagnosis of sporotrichosis was initially suggested by the observation that cross-reactions with paracoccidioidomycosis, cryptococcosis, aspergillosis, candidiasis, and histoplasmosis were absent in the Con-A binding fraction of the cell wall peptide-rhamnomannan (Loureiro Y Penha and Lopes-Bezerra 2000). Later, an ELISA to detect immunoglobulin (Ig)-G in serum from patients with several clinical forms of sporotrichosis was described using this specific antigenic fraction, with a sensitivity of 90 % and specificity of 80 % (Bernardes-Engemann et al. 2005). The Con-A binding fraction of the Sporothrix cell wall peptide-rhamnomannan can also be used to detect antibodies in the synovial fluid (Costa et al. 2008) and to monitor therapeutic response to antifungal treatment (Orofino-Costa et al. 2009).

The exoantigens from Sporothrix spp. were first applied in several immunological assays, such as IEP, LA, and double ID (Karlin and Nielsen 1970; Blumer et al. 1973; Casserone et al. 1983; Albornoz et al. 1984). However, there were no standard procedures for the production of the antigenic extracts used. In fact, changes in sugar composition of the exoantigens occur during Sporothrix growth, indicating a need for standardization (Takata and Ishizaki 1983). Differences in exoantigen composition and immunological reactivity were also related to the morphological state of the fungus (Albornoz et al. 1984), to the culture medium employed (Mendoza et al. 2002; Fernandes et al. 2009), and to the geographical origin of strains (Fernandes et al. 2009). Differences in exoantigen composition among the species of the Sporothrix complex are poorly studied, but it does not appear to significantly impact the antigenic composition of secreted molecules. A recent study showed heterogeneous protein profiles of exoantigens obtained from the mycelial form of S. brasiliensis, S. globosa, and S. schenckii sensu stricto in Sabouraud dextrose medium. The proteins of 60 and 46 kDa were observed in all extracts, regardless of species. Moreover, it was not possible to characterize specific secreted molecules for these species (Fernandes et al. 2013). Similar observations were obtained with the secreted antigens from the yeast form of S. brasiliensis and S. schenckii s. str. A molecule of 85 kDa was detected in both species, and no significant differences in antigenic composition or immunological reactivity were observed among the species (Almeida-Paes et al. 2012).

In a study of the yeast-phase Sporothrix exoantigens, it was verified that a 70-kDa antigenic fraction was always reactive against serum antibodies present in experimentally infected mice after 14 days of infection (Nascimento and Almeida 2005). Further studies against this antigen showed that a monoclonal antibody with affinity to this protein was able to enhance phagocytosis by macrophages. Moreover, this monoclonal antibody reduced the fungal burden and inhibited the Sporothrix interaction with the extracellular matrix (Nascimento et al. 2008). The role of this antigen as an adhesin was elucidated in subsequent studies (Ruiz-Baca et al. 2009; Teixeira et al. 2009). It is interesting to note that an immunological reactivity against the Sporothrix gp-70 was consistently observed in these studies, which encourages its use in the serodiagnosis of sporotrichosis. Furthermore, this antigen is produced by the three major species of the Sporothrix complex (S. brasiliensis, S. globosa, and S. schenckii), thus allowing the sporotrichosis diagnosis regardless of the infective species (Ruiz-Baca et al. 2014).

In general, serological tests as an aid for diagnosis do not use purified or recombinant antigens, because described immune reactive proteins are scarce, especially for the newly described Sporothrix species such as S. brasiliensis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which varied in intensity. An 85-kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used, and the immunodominant protein was identified. However, this protein cross-reacted with sera samples from patients infected with other pathogens (Almeida-Paes et al. 2012). It was also demonstrated that the use of different strains or even the morphological form of Sporothrix isolates could have an effect on the antigenic reactivity of a Sporothrix extract. Therefore, an adequate standardization of antigens must be produced before their general use in the serodiagnosis of sporotrichosis.

8.3 Antibody Detection

8.3.1 Immunoprecipitation and Agglutination Techniques

Immunoprecipitation and agglutination methodologies were first used in the diagnosis of sporotrichosis in the period 1970–1980 (Albornoz et al. 1984; Blumer et al. 1973; Casserone et al. 1983; Karlin and Nielsen 1970). The first TA and CF tests for sporotrichosis were reported in 1910 (Widal et al. 1910), and a diagnostic precipitin test that employed a polysaccharide antigen was subsequently described by González-Ochoa and Figueroa (1947). The ID test for sporotrichosis usually does not cross-react with sera from patients with chromoblastomycosis or leishmaniasis, infectious diseases with similar clinical manifestations (Albornoz et al. 1984). IEP has also been used, and in all positive cases, an anodic arc, called an S arc, is observed (Albornoz et al. 1984). Both methodologies that use an antigenic complex from fungal culture filtrate are highly sensitive. TA and LA both have high sensitivity and specificity and have been used for sporotrichosis serodiagnosis since the 1970s (Blumer et al. 1973; Casserone et al. 1983; Karlin and Nielsen 1970). However, these tests lack sensitivity in cases of cutaneous sporotrichosis (Albornoz et al. 1984; Rippon 1988) and do not enable the determination of the immunoglobulin isotype involved. The lack of standardization of reagents and methodologies applied in these techniques means they are not routinely used in the diagnosis of sporotrichosis in clinical laboratories.

8.3.2 Immunoenzymatic Assays

The serodiagnosis of this mycosis has increasingly used immunoassays. The first immunoblot assay used for diagnosis of sporotrichosis dates back to 1989, when exoantigen preparations from the S. schenckii yeast form showed 100 % sensitivity and 95 % specificity for the detection of antibodies (Scott and Muchmore 1989). Later, another immunoassay (ELISA) was developed, using the Con-A binding peptide-rhamnomannan from the S. schenckii yeast cell wall, and antibodies were detected in 35 serum samples from patients with culture-proven sporotrichosis, resulting in 100 % sensitivity. However, the specificity was lower than previous tests because there was cross-reactivity with sera from patients with cutaneous leishmaniasis (Penha and Lopes-Bezerra 2000). The same group reported on an ELISA test using the same antigenic preparation against sera from 92 patients with different clinical forms of sporotrichosis in Rio de Janeiro and reported 90 % sensitivity, 80 % specificity, and a global efficiency of 86 % (Bernardes-Engemann et al. 2005). Other studies showed that the use of different strains during the preparation of the antigen might result in different sensitivity and specificity, despite the purification of the antigen involved in this methodology. This difference is due to the O-glycan residues linked to the molecules (Bernardes-Engemann et al. 2009).

An ELISA to detect IgG antibodies reactive to the mycelial-phase Sporothrix exoantigens produced after 21 days of growth in Sabouraud dextrose medium at 28 °C showed 97 % sensitivity and 89 % specificity when performed on 90 sera from patients with different clinical forms of sporotrichosis, 72 sera from patients with other infectious diseases, and 76 healthy controls. The major antigenic components present in this preparation were proteins of 90, 70, 63, 51, and 42 kDa (Almeida-Paes et al. 2007a). This assay was further improved to detect IgG, IgM, and IgA antibodies, which improves the global efficiency for diagnosis and therapeutic follow-up of sporotrichosis (Almeida-Paes et al. 2007b). Mendoza et al. (2002) previously described this exoantigen, and the lack of cross-reactivity of this preparation with sera from patients with other mycoses was remarkable. The same antigen was used previously in ID and IEP techniques without cross-reactivity with sera from patients with leishmaniasis or chromoblastomycosis (Albornoz et al. 1984).

When the ELISAs probed with different antigenic preparations are compared, the crude exoantigens (Almeida-Paes et al. 2007a) gave slightly higher sensitivity and specificity than those using the con-A binding fraction of the S. schenckii yeast cell wall (Bernardes-Engemann et al. 2005). A similar observation was found when using this con-A binding fraction and crude exoantigens for the serodiagnosis of feline sporotrichosis. The use of crude exoantigens showed slightly better results than the purified peptide-rhamnomannan in terms of sensitivity (96 and 90 %) and specificity (98 and 96 %, respectively), suggesting that the Sporothrix secreted proteins are highly immunogenic and specific (Fernandes et al. 2011).

More recently, an immunoblot assay using cell-free exoantigens of the yeast form of S. brasiliensis was described to detect IgG antibodies in serum samples from human patients. In this assay, a sensitivity of 100 % was achieved, but a low specificity (50 %) was observed. However, if the authors verified a positive serum sample only if at least two immunological bands appeared in the immunoblots, the specificity increased to 80 % with 93 % sensitivity (Almeida-Paes et al. 2012).

8.3.3 Sporotrichin Skin Test

The skin test (ST) , with mycelial extracts or yeast cells called sporotrichin, has been widely used throughout the world, especially for epidemiological studies of sporotrichosis. It has also been applied as support for diagnosis in Latin America (Lopes-Bezerra et al. 2006; Barros et al. 2011; Bonifaz and Vázquez-González 2013) and in atypical forms of the disease, such as the case of a recent bulbar conjunctival sporotrichosis (Kashima et al. 2010). However, sporotrichin is not available commercially in many countries (Dominguez-Soto and Hojyo-Tomoka 1983; Bonifaz and Vázquez-González 2013), but it is accessible in many institutions dedicated to biomedical research. Reports are contradictory concerning its usefulness as a diagnostic tool due to false-positive results without signs or symptoms of the disease, but this condition suggests a previous immunosensitizing contact (exposure) with S. schenckii. Other reports mention almost 100 % positive sporotrichin ST in lymphocutaneous and fixed cutaneous forms of sporotrichosis (Barros et al. 2011; Bonifaz and Vázquez-González 2010).

The use of sporotrichin ST in epidemiological surveys has been extensively developed from the first surveys in Mexico (González-Ochoa and Ricoy 1970), Guatemala (Mayorga et al. 1978), and Brazil (Rocha-Posada 1968), among others. This practical test has been used until the present (Bonifaz et al. 2013), demonstrating the usefulness of these antigens to identify endemic regions of sporotrichosis worldwide, an option to gain insight in disease emergence scenarios such as the recent cat zoonosis in Brazil (Barros et al. 2004; Oliveira et al. 2011; Freitas et al. 2010; Silva et al. 2012).

Concerning the term sporotrichin, there are reports of different type of antigens. Culture filtrate extracts from the mycelial (28 °C) or yeast (37 °C) forms, commonly called metabolic antigens, were first described by González-Ochoa and Figueroa (1947) and consisted of glycopeptide antigens. Another described and used antigen is a 1:1000, 1:2000, or 1:4000 dilution of heat-killed yeast cells. In addition, their biological properties depend on culture conditions (Takata and Ishizaki 1983; Arenas and Toriello 1986). A commonly used antigen for epidemiological surveys is a culture filtrate of the mycelial form standardized to 10 μg protein/0.1 ml of intradermal ST (Toriello et al. 1991; Bonifaz et al. 2013). The ST may be applied on the back or forearm and readings made at 24 and 48 h. A reaction of 8 mm of induration after 24 and 48 h constitutes a positive test.

The lack of standardization in the above-mentioned antigens would account for the difference in reactivity with sporotrichin ST, such as a 6.25 % positivity with a 1:4000 dilution of a yeast antigen in the southern state of Oaxaca, Mexico with S. schenckii isolates recovered from soil (Sánchez-Aleman et al. 2004). This is in contrast with the 14 % positivity observed with a mycelial metabolic antigen at 10 μg protein/0.1 ml in an endemic region of sporotrichosis in Puebla, Mexico, without any fungal isolation (Mendez-Tovar et al. 2003).

Additional epidemiological studies of sporotrichosis and the S. schenckii complex are necessary because changes in the interplay of pathogens, hosts, and environment lead to the formation of novel disease patterns as observed for sporotrichosis.

8.4 Molecular Diagnosis

Molecular methods have been developed based on polymerase chain reaction (PCR) techniques that show good sensitivity, specificity, and speed for early diagnosis of S. schenckii (Berbee and Taylor 1992; Kano et al. 2001, 2003; Hu et al. 2003; Xu et al. 2010; Mendoza et al. 2012; Liu et al. 2013; Ruiz-Baca et al. 2013; Oliveira et al. 2014). Sandhu et al. (1995) proposed the pioneering DNA-based methodologies used for the diagnosis of fungal infections, and developed 21 specific nucleic acid probes targeting the large subunit of the ribosomal RNA (rRNA) gene from several fungi, including S. schenckii. Among the major genes described for the diagnosis of sporotrichosis are the chitin-synthase gene 1 (CHS1), 18S rDNA, and mitochondrial DNA (mtDNA) (Berbee and Taylor 1992; Kano et al. 2001, 2003; Hu et al. 2003; Xu et al. 2010). The trials with nested PCR using the 18S rDNA gene and mtDNA as targets showed high sensitivity and specificity, indicating that these tests can provide a rapid diagnosis with sufficient accuracy to be used clinically in patients with sporotrichosis (Hu et al. 2003; Xu et al. 2010; Oliveira et al. 2014). A drawback of these methods is that they are not commercially available and require specialized equipment; therefore, they are not carried out in most clinical laboratories.

8.5 Conclusions and Perspectives

Sporotrichosis is classically diagnosed by correlation among clinical, epidemiological, and laboratory data. The conventional method for definitive diagnosis of sporotrichosis is based on etiological agent isolation in culture and its identification. A disadvantage of the culture methods, is that they are difficult to apply in disseminated and/or systemic sporotrichosis; therefore, detection of an antibody response in patients could provide a faster method for diagnosis. Sporotrichin ST remains an option for epidemiological studies to gain insight in disease emergence scenarios such as the recent cat zoonosis in Brazil. To date, serological techniques (like ELISA, Western blot, immunodiffusion, etc.) are used for sporotrichosis diagnosis and involve antibody detection against soluble antigens and different molecules of the S. schenckii cell wall, such as peptide-rhamnomannans, exoantigens, and the newly described molecule gp70. The sensitivity and specificity of these methods differ depending on the antigenic fraction used. Fluorescent antibodies and immunohistochemical techniques are alternative methods that also provide a rapid diagnosis of sporotrichosis in tissue samples. The diagnosis of S. schenckii by PCR in clinical samples has also shown a high degree of sensitivity and specificity. These tests can provide a rapid diagnosis with sufficient precision to be used clinically for patients with sporotrichosis, with PCR studies for the disseminated cases of the disease.

The different methods described in this review are not available in many clinical laboratories, which compromises the ability to diagnose and provide individual treatment for sporotrichosis. The search for new protein biomarkers for the development of an easy, sensitive, and specific methodology could help lower the treatment costs and offer alternatives to current tests for the diagnosis of sporotrichoid infections that can be confused by infections caused by other pathogens. To date, efforts continue to improve or develop new diagnostic tests that are more sensitive and specific for this mycosis. However, these tests require validation prior to general application in routine diagnosis. The development and application of new biomarker molecules for the diagnosis of sporotrichosis remains to be done either with exoantigens or cell wall antigens. The recent release of the S. schenckii genome, and the use of certain tools such as genomics, proteomics, inmunoproteomics, metabolomics, interactomics, etc., would enable major advances in the search and identification of new biomarker antigens for sporotrichosis, which are expected to be introduced to the clinical laboratory in the short or medium term.