Introduction

Subcutaneous mycoses include a heterogeneous group of infection characterized by the development of lesions at the inoculation site. In some diseases, slow extension via lymphatic channels is a frequent occurrence (sporotrichosis); in others, hematogenous and lymphatic dissemination is rarely recorded (chromoblastomycosis) [1].

The diagnosis difficulty of the mycoses is related to several factors, such as subcutaneous lesions macroscopically similar, different etiologic agents, a wide variety of lesions, and their morphologic subtypes [2]. Based on that, immunohistochemical methods have been tested as a support tool for the differential diagnosis of mycoses [3, 4].

In fungus taxonomy, cell wall constitution may be an additional distinguishing criterion. Alongside techniques such as chemical extraction, enzymatic digestion followed by an analysis of hydrolysis products, comparison of antigen fractions using immune sera or by electron microscopy, lectin binding has proved to be a valuable method for the characterization of the cell wall sugar profile [5, 6]. Moreover, lectins interact with carbohydrates, favoring the understanding of pathobiochemistry alterations and progression of tumors originated by mycoses [7, 8].

Furthermore, specific recognition between fungal parasites and their host cell target may be mediated by the interaction of carbohydrate-binding proteins, as lectins on the surface of one type of cell, which combine with complementary sugars on the surface of another cell [9].

Recently, many studies were developed in our laboratories aiming the investigation of different pathologic process [10–12], indicating an important contribution of those macromolecules in this biomedical analysis.

In addition, image analysis system has been used as a support to the interpretation of results obtained from histochemistry techniques, which supplies qualitative and quantitative data related to histomorphology of lesions and anatomy of etiologic agents from skin infections [13–15]. Among advantages of the image analysis, one can include increased precision of measurement of pathological changes; evaluation of effects from different methods of histological processing; determination in the way; and standard sizes for teaching and diagnosis as well as maximization as research tool [11, 16, 17].

The present work aims to investigate the histochemical pattern of subcutaneous lesions in patients with sporotrichosis and chromoblastomycosis and the carbohydrate profile expressed in the cell wall of Sporothrix schenckii Hektoen and Perkins, Fonsecaea pedrosoi (Brumpt) Negroni, Phialophora verrucosa Medlar, and Cladophialophora carrionii (Trejos) using lectin histochemistry.

Materials and Methods

Case Selection

Biopsies from both sexes (mean age 40 years) diagnosed with sporotrichosis (ST; n = 10) and chromoblastomycosis (CM; n = 10), presenting granulomatous lesions, were selected from the Dermatology Service from the University Hospital located in Federal University of Pernambuco (UFPE) and from the Integrated Center of Anatomy and Pathology (CIAP) of the State University of Pernambuco (UPE). The study was approved by the Research Ethics Committee of UFPE, according to the Declaration of Helsinki.

Microorganisms and Growth Conditions

Sporothrix schenckii (URM 2865), Fonsecaeapedrosoi (URM 3161), Phialophoraverrucosa (URM 2884), and Cladophialophoracarrionii (URM 2871) cultures were obtained from URM Culture Collection of Department of Mycology, Biological Sciences Center (UFPE) and have been stocked in mineral oil (Sherf, 1943). Viability test and taxonomic review were carried out by inoculating cultures in test tubes containing 5 ml of 2% dextrose broth [18], under non-stationary conditions, at 28°C, for 30 days. The organisms were indentified by observing macromorphologic (coloration, texture and colony diameter) and micromorphologic (somatic and reproductive structures) characteristics through incubation in potato dextrose agar broth (Difco) [19].

Histochemistry

Histological slices (4 μm) were submitted separately to the following staining techniques hematoxylin and eosin (HE), tricomic of Masson (TM), periodic acid of Shiff (PAS) and Van Gienson (VG) according to Spicer [20].

Lectin Histochemistry

Fungi studied were adhered to glass slides previously treated with 3-amine-propyltriethoxy-silane—APES, (Sigma Chemical Co, USA). Sporothrix schenckii, Fonsecaea pedrosoi, Phialophora verrucosa, and Cladophialophora carrionii, adhered to slides, were treated with a 2% trypsin solution for 1 h at 37°C and a methanol-H2O2 solution for 15 min, followed by incubation with Concanavalin A (Con A, 25 μg/ml) and wheat germ agglutinin (WGA, 25 μg/ml) lectins, both conjugated the peroxidase at 4°C for 3 h. Peroxidase was visualized using diaminobendizine–H2O2 solution for 5–8 min. Samples were counter stained with hematoxylin and evaluated through optical microscopy. Between each step, samples were washed twice (5 min each) with a solution of 0.01 M sodium phosphate buffer, pH 7.2, containing 0.15 m NaCl.

Digital Image Analysis

Digital image analysis (triplicates) was carried out using a digital video camera (Model KT2050-I, Sony, Japan) connected to a microscope (OLYMPUS, Model BH-2, Japan). Images were processed using OPTIMAS™ version 6.1 (Optimas Corporation, USA). All parameters were set according to manufacturer’s instruction. The morphometric parameters evaluated were total medium area (μm2) and number of particles for field captured in the histological slice (in pixels).

Staining control (tissues treated with sugar-inhibited lectins and normal skin samples) was obtained to minimize distortions in values due to the presence of non-marked cells, thus a correction factor (CF) was applied according to the equation CF = s/S, in which s means relative area of the surface and S, the total measured area [10, 12].

In cases which tissues presented total areas with similar and better histomorphologic conditions, the summary of lectin-binding cells per area was developed in three random areas of stained tissues (total area analyzed for each slice = 12,234 μm2).

Lectin-binding patterns were used to calculate (%) the mean area (weak stain = 15–20%; moderate stain = 25–55%; intense stain = 60–95%). Semiquantitative optical analysis was carried out taking into account the intensity of staining pattern indicated as weak (+), moderate (++), or intense (+++), according to Özer [21].

Statistical Analysis

Histochemistry staining patterns and morphometric parameters obtained from digital image analysis were evaluated using t-Student and Tukey analysis (P < 0.05) through the software PRISM™ 3.0.

Results

Results confirmed a large deposition of elastic fibers and collagen (Fig. 1) in the subcutaneous lesions found in sporotrichosis when compared with those observed in chromoblastomycosis. These data are also observed via the mean area of interstitial fibrosis (Table 1). The glycosaminoglycan expression, evidenced by periodic acid of Schiff, was not statistically different in the cellular deposits among studied cases.

Fig. 1
figure 1

a Subcutaneous chromoblastomycosis exhibiting few and short elastic fibers (arrow). b Skin lesion caused by sporotrichosis, exhibiting extensive elastic fibers (arrow). Van Gienson technique. c Interstitial collagen (arrow) in subcutaneous lesion of chromoblastomycosis. d Skin lesion caused by sporotrichosis, exhibiting intense deposits of collagen fibers (arrow). Trichromic of Masson technique. (magnification 400×)

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Table 1 Image analysis of fibrillar components and fibrosis area in patients with subcutaneous mycoses
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Lectin histochemistry evidenced the carbohydrate profile expressed in glycoconjugates of the fungi cell surface, which presented an intense staining pattern in the Sporothrix schenckii cell wall, for both lectins used. This fact suggests that Sporothrix schenckii presents high expression of glucose/mannose and N-acetyl-glucosamine in those cell surface molecules. However, WGA and Con A did not provide evidences for carbohydrate residues in the cell surface of Fonsecaea pedrosoi, Phialophora verrucosa, and Cladophialophora carrionii (Table 2).

Table 2 Lectin histochemistry of fungal cell wall from etiological agents of sporotrichosis and chromoblastomycosis
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Discussion

The fungi usually present intra-and/or extracellular host parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules, per example, presence of sugar-binding proteins (lectins) on the microconidia, suggesting a (glyco)protein nature of the binding sites [7].

The use of lectin histochemistry in mycological studies was first applied in France, in the 1970 decade, and turned out to be a good method for characterizing sugars from the fungal cell wall [22]. A previous study analyzed five species from three distinct genus (Neocallimastix frontalis, N. patriciarum, N. joyonii, Piramonas communis, and Sphaeromonas communis) and the eight different lectins used proved that such technique is an important auxiliary tool to distinguish different fungal species, besides characterizing the carbohydrate nature of the cell wall [5].

Moreover, lectin-binding analysis the cell surface glycoconjugates could be another approach for the identification and typing of medically important fungal species, and it is an indicative of a new and unrecognized mechanism by which the taxonomy may be readily performed in the majority of the laboratories [6].

Thus, Munõz et al. [23] describe lectin profiling for 93 Candida isolates of five different species, using 14 lectins to cover a wide range of sugar specificities, indicating the existence of various biotypes.

Furthermore, information over saccharide profile in fungal surface enables the use of lectins, besides promising cells probes, to be used in therapeutic treatment as already proposed for bacterial diseases [24]. These proteins might serve as carriers of antifungal agents, using as targets their specific carbohydrates found in the cell surface of microorganisms.

According to clinical and histological data [1, 25], subcutaneous lesions, in sporotrichosis and chromoblastomycosis, present quite similar morphologic characteristics (granulomatous lesions), which in most of the cases, it is necessary to isolate etiological agents and therefore to define sporotrichosis or chromoblastomycosis diagnosis. Studies aiming the elucidation of such questions are scarce.

In certain persistent or non-degraded antigens, commonly found in extracellular matrix associated with fungal cell wall, initial lymphocytic infiltration is substituted by macrophages. These mononuclear cells suffer modifications in the chronic disease. In chromoblastomycosis, it was also observed massive pseudoepitheliomatous hyperplasia with numerous small abscesses and brown sclerotic bodies of fungus within giant cell [26]. In addition, the histological lesions, caused by sporotrichosis, possess an absence of organisms in biopsy material, but present widespread fibrillar components deposition in the interstice, which demonstrates to be the main cause of the intense fibrosis [2].

Our study suggests a differentiated composition of carbohydrates in the cell wall of the fungi studied [6]. That would influence the distinct processes of interaction of the etiological agents of both mycosis with the host defense cells during the infection, since the hypersensitivity to etiological agent of fungal origin is type IV and so it is related to glycoproteins present in the cell wall [27–29].

Limongi et al. [30] showed previously that mannose and N-acetylglucosamine (GlcNAc) residues are involved in the process of adhesion of Fonsecaea pedrosoi, the causative agent of chromoblastomycosis, to epithelial cells. It was then suggested that lectin-like molecules would be involved in the interaction.

Expression of different glycoconjugates leads toward a differentiated immune response [28]. Our results indicated the absence or non-accessibility to residues of glucose and/or mannose and N-acetyl-glucosamine in glycoconjugates in the cell surface of Fonsecaea pedrosoi, Phialophora verrucosa, and Cladophialophora carrionii. Failure in recognizing such carbohydrates may be explained based on the fact that since Con A and WGA are not endolectins, lectins are able to bind internal saccharide residues of the glyco-structure of glycoconjugates, such sugars were not reached by both lectins. On the other hand, such molecules possess the ability to bond to glucose and/or mannose and N-acetyl-glucosamine, respectively, at a non-reducing terminal of the saccharide chain. Furthermore, several studies suggested that carbohydrate expression varies according to the age and environmental conditions of the fungus [31].

In addition, Sidrim and Rocha [25] cited that in pathogenic fungi, a few reports suggest the occurrence of sialic acids in C. neoformans, Sporothrix schenckii, Fonsecaea pedrosoi, and Paracoccidioides brasiliensis. On the other hand, both N-acetyl and N-glycolyl derivatives were reported in S. schenckii and F. pedrosoi. In S. schenckii, sialic acids protect yeast forms against phagocytosis, and in F. pedrosoi, neuraminic acid derivatives were associated with morphogenesis and cellular integrity.

Our results are in agreement with those few studies that used lectin histochemistry in order to investigate the correlation between cell surface glycoconjugates and tissue inflammatory response to the fungi as Sporothrix schenckii [9, 32–34].

According to Esquenazi and coworkers [7], lectin–carbohydrate interaction was shown to play a crucial role in the infectious process. Many investigations recognized that all cells are coated with sugars and that many also express surface lectins. Since epithelial cells may express these carbohydrates, the next step will be to investigate the possible role of these carbohydrates on the cellular interaction using skin tissues from dermatophytosis.

It is possible to conclude that even if subcutaneous lesions related to sporotrichosis and chromoblastomycosis present similar morphologic characteristics, both conditions present different histopathologic profiles regarding to deposit of collagen, elastic fibers, and level of interstitial fibrosis. The expression of carbohydrate profile in the cell surface was observed to be different and characteristic to the species evaluated using lectin histochemistry.