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Feline sporotrichosis: Histopathological profile of cutaneous lesions and their correlation with clinical presentation
Comparative Immunology, Microbiology and Infectious Diseases 36 (2013) 425–432
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Feline sporotrichosis: Histopathological profile of cutaneous lesions and their correlation with clinical presentation Luisa H.M. Miranda a,∗ , Fátima Conceic¸ão-Silva b , Leonardo P. Quintella c , Bianca P. Kuraiem a , Sandro A. Pereira a , Tânia M.P. Schubach a a Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos, Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Fundac¸ão Oswaldo Cruz (FIOCRUZ), Av. Brasil, 4365, Rio de Janeiro 21040-360, Brazil b Laboratório de Imunoparasitologia, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Leônidas Deane, Av. Brasil, 4365, Rio de Janeiro 21040-360, Brazil c Servic¸o de Anatomia Patológica, IPEC, FIOCRUZ, Pavilhão Gaspar Viana, Av. Brasil, 4365, Rio de Janeiro 21040-360, Brazil
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Article history: Received 30 August 2012 Received in revised form 23 January 2013 Accepted 30 March 2013 Keywords: Cat Sporotrichosis Histopathology Skin Sporothrix schenckii
a b s t r a c t Cutaneous lesions of feline sporotrichosis show high fungal load and are associated with severe disease and elevated zoonotic potential. The present study describes the histopathology and fungal load of the lesions in different clinical presentations of feline sporotrichosis. Cats with sporotrichosis were separated into groups L1, L2 and L3 (lesions in one, two and three or more locations, respectively) and subjected to skin biopsies for histopathology. Eighty-six cats were included in the study. Lesions were suppurative granulomatous in 84 cases and poorly formed granulomas were predominant. The well-formed granulomas were associated with group L1. The high fungal load was predominant in group L3 and in poorly formed granuloma cases and did not occur in well-formed granulomas cases. The good general condition was associated with low fungal load. These findings suggest that the fungal load control in animals with more localized lesions and well-organized response is linked with the improvement in the outcome of infected cats. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Sporotrichosis is a mycosis that affects humans and many animals worldwide, and is caused by Sporothrix schenckii complex species [1]. Transmission occurs through a traumatic inoculation through the skin with soil, plants, and organic matter contaminated with the fungus [2]. Zoonotic transmission may also occur through a bite, scratch or contact with the exudate from infected lesions. Cats have a high zoonotic potential, since their skin lesions contain plenty of yeast [3]. Since 1998, sporotrichosis has appeared as an epidemic disease in Rio de Janeiro, Brazil, and by 2009, there have been 2200 human cases, 3244 feline cases and 120 canine
∗ Corresponding author. Tel.: +55 21 38659536; fax: +55 21 38659553. E-mail address: [email protected] (L.H.M. Miranda). 0147-9571/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cimid.2013.03.005
cases diagnosed by the Evandro Chagas Clinical Research Institute (IPEC) – FIOCRUZ [4]. Among the human cases reported up till 2004, 83% have reported contact with sporotrichosis infected cats, and of these, 56% resulted from a scratch or bite of the animal [5]. Zoonotic transmission appears to explain the implementation of the epidemic and its maintenance in an endemic form [6]. Several species of the S. schenckii complex have been isolated from human and feline lesions in Rio de Janeiro, Brazil and Sporothrix brasiliensis has been considered the most prevalent species in this current epidemic [4,7], with a prevalence of more than 90.0% of cases. S. schenckii has also been described in few cases (6.0%). Sporothrix mexicana and Sporothrix globosa were observed in a single case by each one [8]. Currently, the cat is the animal species most affected by sporotrichosis [9] and their cutaneous lesions are characterized by subcutaneous nodules which develop into
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ulceration. The clinical signs in feline sporotrichosis are variable, with subclinical infections, single lesions, and fatal disseminated systemic forms [3,10]. Experimental studies in mice suggest that the histopathologic lesions of sporotrichosis vary with the stage of disease, showing first the formation of abscesses and the participation of macrophages and lymphocytes, associated with a high fungal load. When the macrophages activate and begin to differentiate, thereby forming granulomas, the fungal load decreases, accompanied by the reduction of abscesses and increase in plasma cells [11–13]. The occurrence of severe sporotrichosis, including skin lesions without granulomas and rich in fungal elements in cats that are apparently immunocompetent [3,10] show the high susceptibility of these animals to infection by S. schenckii and emphasize the need to better understand the immune response against this fungus and its association with the fungal load and general condition of the animals. The acquired results could help in the design of new drugs and in the treatment and control of feline sporotrichosis, further reducing its transmission to humans and other cats. This present study aims to describe a histopathological profile of feline sporotrichosis lesions and to verify the differences in pathological expression and fungal load, according to clinical presentation, age and general condition of the animals.
of 0.4 cm, after sedation and local anesthesia. The biopsy specimens were fixed in 10% buffered formalin for histological examination at the Department of Pathology, IPEC – FIOCRUZ. The procedures performed on the cats and the use and storage of their biological samples, were approved by the Animal Ethics Committee (CEUA), FIOCRUZ. License number LW-28/10. 2.2. Histopathology
2. Material and methods
Sections (5 ) were stained with haematoxylin and eosin (HE) and Grocott silver stain for microscopic analysis. Histopathological analysis was performed in an optical microscope by a trained technician. The lesions were described considering the type of inflammation – granulomatous or nonspecific. In cases of granulomatous inflammation, the cell activation degree of the mononuclear phagocytic system in granulomas – macrophages or epithelioid cells – and organization of the granuloma were also verified. Table 1 is adapted from a study of Miranda et al. [15] and represents definitions of the histopathological variables used in this study. In the Grocott silver stain analysis, the cases in which yeast-like forms (YLF) were present were considered positive. The cases were considered negative when no YLF was observed after the analysis of 50 high power microscopic fields (400×).
2.1. Sampling
2.3. Statistical analysis
The study was cross-sectional and samples were obtained from outpatient routines at the Domestic Animal Dermatozoonosis Laboratory of Clinical Research (LAPCLIN/DERMZOO) IPEC – FIOCRUZ, during the period of July 2009–August 2011. The animals considered eligible were cats with sporotrichosis (confirmed by isolation and identification of Sporothrix sp. complex in culture medium), showing ulcerated skin lesions, with no previous antifungal or corticosteroid therapy. Cases were excluded when the biopsy sample was considered superficial (not including at least the reticular dermis) or not representative (not exhibiting inflammatory infiltration covering more than one third of the sample). The general condition of the animals was classified as good, fair or poor. Good general condition was considered with the absence of extra-cutaneous signs (0). The presence of signs such as dyspnea, conjunctivitis, weight loss, dehydration, pale mucous membranes and prostration, when mild, indicate fair general condition (1 or 2), and when accented, poor general condition (3 or 4). The cats were divided into three groups according to the distribution of skin lesions: L1, L2 and L3 [3]. In L1, cats with lesions in one location were included; in L2, cats with lesions in two non-contiguous locations were included, and in L3, cats with lesions in three or more non-contiguous locations were included. Samples were obtained from exudates of skin ulcers by means of sterile swabs (isolation and identification of fungi in culture medium) [14] and from fragments of the edge of the ulcerated lesion by means of biopsy, using a punch
Data were stored and analyzed with the Statistical Package for Social Sciences (SPSS) for Windows, version 16.0 (SPSS Inc.). For descriptive analysis, we calculated the frequencies of the variables. The different groups were compared regarding the clinical findings and histopathological analysis. The chi-square test of independence was used to determine a significant association between the variables studied. Fisher’s exact test was applied to the comparison of variables with only two categories. In all specified analyses, a p-value < 0.05 was considered as the indication of a statistically significant association. 3. Results 3.1. Sampling One hundred and nine cats, seventy-nine males and thirty females, were observed from July 2009 to August 2011 and eighty-six were included in the study. Seventeen cats were not included due to previous anti-fungal or corticosteroid therapy. One cat was excluded for no isolation of fungi and five because biopsy samples were considered non-representative or superficial. 3.2. Histopathological findings Microscopically, 84 (97.7%) showed suppurative granulomatous inflammation and two (2.3%) showed nonspecific inflammation.
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Table 1 Terms and definitions used in the histopathological classification of feline sporotrichosis. Histopathological term Granuloma/granulomatous inflammation
Categories
Definition Inflammatory infiltrates contain activated cells of the mononuclear phagocyte system (MPS) forming compact aggregates or interstitial cords
Nonspecific inflammation
Inflammatory infiltrates without granuloma
Suppurative granuloma
Granuloma with neutrophil infiltration
Fungal granuloma
Granulomas in which macrophages are filled with yeast-like forms throughout the length of the granuloma. Reaction lympho-plasmacytic scarce Mild to moderate
Intensity of neutrophils infiltration of a granuloma
Marked Mild to moderate
Intensity of lymphocytes infiltration of a granuloma
Marked Mild to moderate
Intensity of plasma cells infiltration of a granuloma
Marked
Activated cells of the MPS in granulomas
Macrophages Epithelioid cells
Granuloma organization
Well formed Poorly formed
Infiltration of neutrophils into the granuloma is scattered, in loose and small clusters Infiltration of neutrophils into the granuloma is dense and more cellular than that of mild to moderate intensity infiltrates Infiltration of lymphocytes into the granuloma is scattered, in loose and small clusters Infiltration of lymphocytes into the granuloma is dense and more cellular than that of mild to moderate intensity infiltrates Infiltration of plasma cells into the granuloma is scattered, in loose and small clusters Infiltration of plasma cells into the granuloma is dense and more cellular than that of mild to moderate intensity infiltrates Large polygonal cells with plump eccentric nuclei and pale eosinophilic, slightly granular, cytoplasm with distinct borders Large elongate cells with eccentric reniform nuclei and abundant pale eosinophilic cytoplasm with indistinct borders Well delineated with a predominance of epithelioid cells Poorly delineated and/or a predominance of macrophages
MPS: mononuclear phagocytic system.
3.2.1. Suppurative granulomatous inflammation (n = 84) Eighty-four cases were characterized by suppurative granulomatous inflammation: 12 (14.3%) from group L1, 15 (17.9%) from L2 and 57 (67.9%) from L3. Table 2 shows the frequencies of the clinic-epidemiological variables for the cases characterized by suppurative granulomatous inflammation. The infection was predominant in males from all three groups, with significant difference in the L3 group (pvalue = 0.006). Among the cats of L1 (n = 12), eight were in good general condition and none of the cats included in groups L1 and L2 had poor general condition. Table 3 shows the frequencies of histopathological findings from each group. The proportion of poorly formed granulomas (Fig. 1A) was different amongst the groups:
Table 2 Feline sporotrichosis: frequencies of the clinical-epidemiological variables in cases characterized by suppurative granulomatous inflammation. Variable (n)
Lesions characterized by suppurative granulomatous inflammation (N = 84) n (%)
General condition (84)
Good Fair Poor
41 (48.8) 27 (36.1) 16 (19.1)
Age (79)
>0 < 2 years ≥2 < 5 years ≥5 years
26 (32.9) 43 (54.4) 10 (12.7)
Gender (84)
Male Female
62 (73.8) 22 (26.2)
75.0% in group L1, 80.0% in L2 and 94.6% in L3 (pvalue = 0.048). Fungal granulomas were observed in 34 cases (41.7%): 8.3% of cases from group L1, 26.7% from L2 and 51.8% from L3 (p-value = 0.008). There was a significant difference (p-value = 0.004) regarding the fungal granuloma and the general condition: among the 41 cats in good general condition, nine (22.0%) had fungal granulomas. In cats with fair and poor general condition, this percentage was 55.6% and 62.5%, respectively. In cats over five years of age (n = 10), the presence of epithelioid cells and well formed granulomas (Fig. 1B) had a percentage of 50.0% and 42.9% respectively. In cats of less than two and of two to five years aged, the presence of epithelioid cells had percentage of 8.0% and 11.6%, respectively (p = 0.005) and the well formed granulomas had percentage of 4.0% and 9.3%, respectively. The intensity of neutrophils was marked (Fig. 1C) in 55 (65.5%) cases: 58.2% in cats in good general condition, 29.1% in cats with fair general condition and 12.7% in cats with poor general condition. Among the cats in good general condition (n = 41), 32 (80.0%) exhibited marked intensity of neutrophils (p-value = 0.022). Among the cases with marked intensity of lymphocytes (n = 30), 66.7% had good general condition, 26.6% had fair general condition and 6.7% had poor general condition (p-value = 0.021). Among the cases with mild to moderate lymphocyte (n = 54), 22.4% were less than two years old, 69.4% were two to five years old and 8.2% were over five years old (pvalue = 0.004).
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Table 3 Feline sporotrichosis: frequencies of each histopathological finding in cases characterized by suppurative granulomatous inflammation in three groups. Histopathological finding
Clinical presentation L1 (N = 12) n (%)
Total L2 (N = 15) n (%)
L3 (N = 57) n (%)
n (%)
Granuloma organization
Poorly formed Well formed
9 (75.0) 3 (25.0)
12 (80.0) 3 (20.0)
54 (94.7) 3 (5.3)
75 (89.3) 9 (10.7)
Predominant activated cells of the MPS in granulomas
Macrophage Epithelioid cells
9 (75.0) 3 (25.0)
11 (73.3) 4 (26.7)
51 (89.5) 6 (10.5)
71 (84.5) 13 (15.5)
Fungal granuloma
Present Absent
1 (8.3) 11 (91.7)
4 (26.7) 11 (73.3)
29 (50.9) 28 (49.1)
34 (40.5) 50 (59.5)
Neutrophils infiltrate intensity
Mild to moderate Marked
1 (8.3) 11 (91.7)
5 (33.3) 10 (66.7)
23 (40.4) 34 (59.6)
29 (34.5) 55 (65.5)
Lymphocytes infiltrate intensity
Mild to moderate Marked
6 (50.0) 6 (50.0)
8 (53.3) 7 (46.7)
40 (70.2) 17 (29.8)
54 (64.3) 30 (35.7)
Plasma cells infiltrate intensity
Absent Mild to moderate Marked
0 4 (33.3) 8 (66.7)
1 (6.7) 7 (46.7) 7 (46.7)
0 28 (49.1) 29 (50.9)
1 (1.2) 39 (46.4) 44 (52.4)
L1: cats with lesions in one location; L2: cats with lesions in two non-contiguous locations; L3: cats with lesions in three or more non-contiguous locations. MPS: mononuclear phagocytic system.
3.2.2. Nonspecific inflammation (n = 2) Two cases showed no granulomas on histopathological analysis: one from the L1 group and another from the L2. Both were male and in good condition. One of them was less than two years old and the other was over five. The nonspecific inflammation showed marked intensity of neutrophils in both cases. The lymphocytes infiltration showed marked intensity in one case and mild to
moderate in the other. Plasma cells showed mild to moderate intensity in both cases. 3.2.3. Grocott silver stain In cases characterized by suppurative granulomatous inflammation (n = 84), the Grocott silver stain revealed YLF in 79 cases (94.0%): 11 from group L1, 12 from L2 and 56 from L3 (p-value = 0.027).
Fig. 1. Feline sporotrichosis. Skin. (A) Fungal granuloma. Poorly formed suppurative granuloma, in which macrophages are filled with yeast-like forms. Hematoxyline-Eosine. (B) Well-formed suppurative granuloma. Hematoxyline-Eosine. (C) Marked intensity of neutrophils. Hematoxyline-Eosine. (D) High fungal load. Grocott silver stain.
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Table 4 Feline sporotrichosis: evaluation of fungal load in the histological sections by the technique of Grocott silver stain regarding the clinical and epidemiological findings. Variable (n)
Fungal load at GSS Absent
Up to 100 yeast-like forms/field*
Over 100 yeast-like forms/field*
General condition n (%)
Good (41) Fair (27) Poor (16)
4 (9.7) 1 (3.7) 0
17 (41.5) 9 (33.3) 3 (18.8)
20 (48.8) 17 (63.0) 13 (81.2)
Age n (%)
>0 < 2 years (26) ≥2 < 5 years (43) ≥5 years (10)
0 2 (4.6) 2 (20.0)
12 (46.2) 11 (25.6) 4 (40.0)
14 (53.8) 30 (69.8) 4 (40.0)
Gender n (%)
Male (62) Female (22)
4 (6.4) 1 (4.5)
20 (32.3) 9 (40.9)
38 (61.3) 12 (54.6)
GSS: Grocott silver stain. * Microscope field of 400×.
In 45 (53.6%) cases, more than 400 YLF/field of 400× (Fig. 1D) were observed. This fungal load was observed in 25.0% of cases from group L1, 40.0% from L2 and 63.2% from L3. Tables 4 and 5 show the values of fungal load regarding the clinical, epidemiological and pathological data. In well-formed granulomas, a fungal load of greater than 100 YLF/field of 400× was not observed, in poorly formed granulomas, this fungal load was seen in 66.7% of cases (p-value = 0.001). In cases where granulomas showed a predominance of epithelioid cells, the fungal load exceeding 100 YLF/field was not observed. In granulomas with predominance of macrophages, this load was observed in 72.5% (p-value < 0.001) of cases. Among the cases that showed mild to moderate intensity of neutrophils infiltrate (n = 29), four (14.3%) had fungal load up to 100 YLF/field and 24 (82.1%) load of more than 100 YLF/field. Among the cases showing marked intensity (n = 55), four (7.3%) showed no YLF, 24 (43.6%) had fungal load up to 100 YLF/field and 27 (49.1%) had load higher than 100 YLF/field (p-value = 0.006). In both cases characterized by nonspecific inflammation, the Grocott silver stain showed YLF. One showed
fungal load up to 100 YLF/field and in the other, over 100 YLF/field. 4. Discussion In this study, lesions in multiple locations were prevalent, although half of the cats (50.0%) were in good general condition. This observation was previously described by Schubach et al. [16] and Pereira et al. [9]. In this study, none of cats in groups L1 and L2 had poor general condition. However, the involvement of systemic sporotrichosis by cats without the presence of clinical signs is common [16]. In all groups, there was predominance of males, and the percentage difference in relation to females was significant in the L3 group. The cutaneous lesions of feline sporotrichosis in this study were predominantly suppurative granulomatous and with poorly formed granulomas. Other authors have also described lesions of feline sporotrichosis as suppurative granulomatous [17,18]. However, in a previous study conducted in Rio de Janeiro, the presence of granulomas was not frequent (12.2%) [3,19]. There were no significant differences regarding the presence of granuloma and the different clinical presentations. In addition to the low
Table 5 Feline sporotrichosis: evaluation of fungal load in the histological sections by the technique of Grocott silver stain regarding the histopathologic findings. Histological finding (n)
Fungal load at GSS Absent
Up to 100 yeast-like forms/field*
Over 100 yeast-like forms/field*
Granuloma organization n (%)
Well-formed (9) Poorly formed (75)
3 (33.3) 2 (2.7)
6 (66.7) 22 (29.3)
0 51 (68.0)
Predominant MPS cells n (%)
Epithelioid cells (13) Macrophages (71)
4 (30.8) 1 (1.4)
9 (69.2) 19 (26.8)
0 51 (71.8)
Neutrophils infiltrate intensity n (%)
Mild to moderate (29) Marked (55)
1 (3.4) 4 (7.3)
4 (13.8) 24 (43.6)
24 (82.8) 27 (49.1)
Lymphocytes infiltrate intensity n (%)
Mild to moderate (54) Marked (30)
3 (5.6) 2 (6.7)
16 (29.6) 12 (40.0)
35 (64.8) 16 (53.3)
Plasma cells infiltrate intensity n (%)
Absent (1) Mild to moderate (39) Marked (44)
0 3 (7.7) 2 (4.5)
1 (100.0) 11 (28.2) 16 (36.4)
0 25 (64.1) 26 (59.1)
GSS: Grocott silver stain. MPS: mononuclear phagocytic system. * Microscope field of 400×.
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frequency of granuloma, the percentage of cats in Group L1 (32.8%) was higher [3] compared to that described in this present study (14.3%). This suggests that the tendency to formulate the more organized response described regarding the animals of group L1 was probably not observed before. The granulomas of human and canine sporotrichosis lesions are also predominantly poorly formed [20,21]. However, in dogs and humans, poorly formed granulomas usually show a predominance of epithelioid cells, while the poorly formed granulomas of the feline sporotrichosis had a predominance of macrophages in almost all the lesions described. Additionally, the high frequency of fungal granulomas in feline sporotrichosis reveals the minimal lympho-plasmacytic reaction, normally present in lesions of human and canine sporotrichosis. Marques et al. [22] described similar findings for feline sporotrichosis: a granulomatous infiltration composed primarily of macrophages full of fungal structures, and minimal lympho-plasmacytic reaction In experimental studies of sporotrichosis lesions in mice, the influx of mononuclear phagocytic system cells, their activation and subsequent differentiation into epithelioid cells occur along with the reduction of fungal load and the increase of lymphocytes and plasma cells infiltrated [10,11,23]. Thus, the predominance of macrophages in granulomas in lesions of feline sporotrichosis and the lack of a lympho-plasmacytic reaction may be a reflection of the susceptibility of cats to the fungus and may be related to the high fungal load observed in most cases. The presence of well-formed granulomas and epithelioid cells granulomas in cats over five years old was significant. It was also observed that the intensity of lymphocytes was related to the cat’s age, with smaller proportions of mild to moderate intensity in cats over five years old. The presence of poorly formed granulomas was associated with group L3 and the marked intensity of lymphocytes was associated with good general condition. Together, these findings suggest that cats over five years may have become more resistant to disease than the cats of other age groups. Sporotrichosis lesions in cats generally exhibit a lot of yeast forms. In this study, the detection of fungi was obtained in 79 (94.0%) cases by means of the Grocott silver stain technique, and the highest percentage was observed in lesions of cats from the L3 group. In 52.3% of the cases, more than 400 YLF/field (400×) were identified. This percentage was also higher in cats from group L3, indicating an association between fungal load and the clinical presentation of the animal. The fungal load is directly associated with the intensity of neutrophils in experimental and human sporotrichosis. In human sporotrichosis, the marked intensity of neutrophils is associated with patients with more lesions and longer treatment [24]. However, in this present study, we observed an inverse relationship between intensity of neutrophils and fungal load, with a high proportion of cases presenting high fungal load along with a mild to moderate intensity of neutrophils. Similar observations were described by Hachisuka and Sasai [25] in an experimental study, where immunosuppressed mice exhibited lesions
rich in fungi and with a scarcity of neutrophils and lymphocytes. Kajiwara et al. [26] attributed neutrophils to the involvement in the response of the fungus in mice. The greater efficiency of fungus phagocytosis by neutrophils in these animals could justify a lower fungal load in lesions with infiltrates rich in neutrophils. These results may also show different stages of inflammation: in immunocompetent patients, an increased influx of inflammatory cells in response to a high fungal load. In immunosuppressed patients, the highest concentration of fungal elements may occur by longitudinal growth due to lack of response. Previous studies during the epidemic in Rio de Janeiro also described an inverse relationship between fungal load and granuloma formation in the lesions of feline sporotrichosis [3,19]. However, the sensitivity of the histological method for detection of fungi and the frequency of granuloma were lower than those found in this study. In humans and dogs, the occurrence of severe forms of the disease seems related to immunosuppressive factors such as AIDS [27–30]. In cats, there was no further association between severity of sporotrichosis or fungal load of cutaneous lesions and infection by feline immunodeficiency virus and/or by feline leukemia virus [3,8,31]. Some experimental studies show that the more severe clinical presentations may be associated with more virulent strains of the fungus [32,33]. More virulent strains of S. schenckii induce a weak inflammatory response, facilitating the growth of fungus in the lesion [34]. In the present study, the identification of species of S. schenckii complex was not performed. However, since S. brasiliensis has been pointed as the major prevalent species in Rio de Janeiro, we can suggest that the different conditions observed in the groups L1, L2 and L3 may be related to factors other than the infective species. In addition, it should be noted that in this zoonotic epidemic, the same isolates of the fungi circulate between humans and cats [35], with severe forms of sporotrichosis observed frequently in cats, but not in humans, in which are usually associated with immunosuppressive or concomitant disease factors. This suggests that, besides the virulence of the fungus and the species involved, other factors enable the occurrence of severe disease in cats, despite the absence of immunosuppressive factors and the infective species. Galhardo et al. [36] described a method of molecular fingerprinting to identify the genotypes of S. schenckii isolates recovered from different clinical forms of human sporotrichosis in the epidemic in Rio de Janeiro. According to this study, there was no correlation between the subtypes generated from the strains and the clinical forms. The authors suggest that the main factor determining these clinical forms is probably related to the immune response of the patient. The results suggest a relationship between a well organized immune response (well-formed granulomas and presence of epithelioid cells) and control of fungal load, as well as its association with the general condition of the animal and clinical presentation of the disease. However, further studies are necessary to state whether the different clinical presentations of feline sporotrichosis, fungal load and inflammatory profile are somehow related to infection
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by different species of S. schenckii complex or to different susceptibilities of cats. In addition, studies regarding the immune response of feline sporotrichosis and the disease course in cats could contribute to the better understanding of its pathogenesis and whether the different clinical presentations and inflammatory profiles could be determinant to the prognosis of the disease. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors thank Isabella D.F. Gremião, Marcos F.L. de Almeida, Maria D.P.E. Espinoza, Francisco C.C. Rodrigues, Antonio C. da Silva, Patricia F. Pereira, Luiz C. Ferreira, Rosani S. Reis, Rodrigo C. Menezes, Amanda A.B. Kitada, Érica G. Reis and Elaine W. Souza for technical support. This study was partially financed by the Programa de Apoio a Pesquisa Estratégica em Saúde (PAPES V), FIOCRUZ, by the Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and by the Conselho Nacional de Desenvolvimento Científico Tecnológico (CNPq), Brazil. TMPS is a CNPq researcher. References [1] López-Romero E, Reyes-Montes Mdel R, Pérez-Torres A, RuizBaca E, Villagómez-Castro JC, et al. Sporothrix schenckii complex and sporotrichosis, an emerging health problem. Future Microbiol 2011;6(1):85–102. [2] Rippon J. Sporotrichosis. Journal of Veterinary Internal Medicine 1988;7:44–5. [3] Schubach TMP, Schubach AO, Okamoto T, Barros MB, Figueiredo FB, Cuzzi T, et al. Evaluation of an epidemic of sporotrichosis in cats: 347 cases (1998–2001). Journal of the American Veterinary Medical Association 2004;224:1623–9. [4] Barros MB, de Almeida Paes R, Schubach AO. Sporothrix schenckii and sporotrichosis. Clinical Microbiology Reviews 2011;24(4):633–54. [5] Schubach AO, Barros MBL, Wanke B. Epidemic sporotrichosis. Current Opinions in Infectious Diseases (Philadelphia, PA) 2008;21:129–33. [6] Barros MBL, Schubach TP, Coll JO, Gremião ID, Wanke B, Schubach A, et al. Esporotricose: a evoluc¸ão e os desafios de uma epidemia. Revista Panamericana de Salud Publica 2010;227(6):455–60. [7] Marimon R, Cano J, Gené J, Sutton DA, Kawasaki M, Guarro J. Sporothrix brasiliensis, S. globosa, and S. mexicana, three new Sporothrix species of clinical interest. Journal of Clinical Microbiology 2007;45(10):3198–206. [8] Oliveira MM, Almeida-Paes R, Muniz MM, Gutierrez-Galhardo MC, Zancopé-Oliveira RM. Phenotypic and molecular identification of Sporothrix isolates from an epidemic area of sporotrichosis in Brazil. Mycopathologia 2011;172(4):257–67. [9] Pereira SA, Passos SRL, Silva JN, Gremião IDF, Figueiredo FB, Teixeira JL, et al. Response to azolic antifungal agents for treating feline sporotrichosis. Veterinary Record 2010;166(10):290–4. [10] Schubach TMP, Schubach AO, Cuzzi-Maya T, Okamoto T, Reis RS, Monteiro PCF, et al. Pathology of sporotrichosis in 10 cats in Rio de Janeiro. Veterinary Record 2003;152:172–5. [11] Miyaji M, Nishimura K. Defensive role of granuloma against Sporothrix schenckii infection. Mycopathologia 1982;80:117–24. [12] Mohri S. Study in sporotrichosis – III. Histological and Immunohistochemical study in experimental cutaneous sporotrichosis in man. Yokohama Medical Bulletin 1987;38(1–2):37–48. [13] Hiruma M, Yamaji K, Shimizu H, Ohata H, Kukita A. Ultrastructural study of tissue reaction of mice against Sporothrix schenckii infection. Archives for Dermatological Research 1988;280(Suppl.):94–100.
431
[14] Kwon-Chung K, Bennett J. Sporotrichosis. In: Kwon-Chung K, Bennett J, editors. Medical Mycology – The Pathogenic Fungi and the Pathogenic Actinomycetes. Philadelphia: Lea & Febiger; 1992. p. 707–29. [15] Miranda LHM, Quintella LP, Santos IB, Oliveira RVC, Menezes RC, Figueiredo FB, et al. Comparative histopathological study of sporotrichosis and American tegumentary leishmaniosis in dogs from Rio de Janeiro. Journal of Comparative Pathology 2010;143(1): 1–7. [16] Schubach TMP, Schubach AO, Okamoto T, Pellon IV, Monteiro PCF, Reis RS, et al. Haematogenous spread of Sporothrix schenckii in cats with naturally acquired sporotrichosis. Journal of Small Animal Practice 2003;44:395–8. [17] Dunstan RW, Langham RF, Reinman KA, Wakenell OS. Feline sporotrichosis: a report of five cases with transmission to humans. Journal of the American Academy of Dermatology 1986;15: 37–45. [18] Gonzalez Cabo JF, Heras Guillamon M, Latre Cequiel MV, Garcia de Jalon Ciercoles JA. Feline sporotrichosis: a case report. Mycopathologia 1989;108:149–54. [19] Schubach TMP, Schubach AO, Reis RS, Cuzzi-Maya T, Blanco TCM, Monteiro DF, et al. Sporothrix schenckii isolated from domestic cats with and without sporotrichosis in Rio de Janeiro, Brazil. Mycopathologia 2002;153:83–6. [20] Miranda LHM, Quintella LP, Santos IB, Menezes RC, Figueiredo FB, Gremião IDF, et al. Histopathology of canine sporotrichosis: a morphological study of 86 cases from Rio de Janeiro (2001–2007). Mycopathologia 2009;168(2):79–87. [21] Quintella LP, Passos SR, do Vale AC, Galhardo MC, Barros MB, Cuzzi T, et al. Histopathology of cutaneous sporotrichosis in Rio de Janeiro: a series of 119 consecutive cases. Journal of Cutaneous Pathology 2011;38(1):25–32. [22] Marques SA, Franco SRVS, Camargo RMP, Dias LDF, Haddad Jr V, Fabris VE. Esporotricose do gato doméstico (Felis catus): transmissão humana. Revista do Instituto de Medicina Tropical de São Paulo 1993;35(4):327–30. [23] Peng-Cheng L, Yoshiike T, Yaguchi H, Ogawa H. Histopathological studies of Sporothrix schenckii-inoculated mice – possible functions of polymorphonuclear leukocytes in normal and immunocompromised (congenitally athymic nude) mice. Mycopathologia 1993;122: 89–93. [24] Morgado FN, Schubach AO, Barros MBL, Conceic¸ão-Silva F. The in situ inflamatory profile of lymphocutaneus and fixed forms of human sporotrichosis. Medical Mycology 2011;49(6):612–20. [25] Hachisuka H, Sasai Y. Development of experimental sporotrichosis in normal and modified animals. Mycopathologia 1981;76: 79–82. [26] Kajiwara H, Saito M, Ohga S, Uenotsuchi T, Yoshida S. Impaired host defense against Sporothrix schenckii in mice with chronic granulomatous disease. Infection and Immunity 2004;72(9): 5073–9. [27] Rocha MM, Dassin T, Lira R, Lima EL, Severo LC, Londero AT. Sporotrichosis in a patient with AIDS: report of a case and review. Revista iberoamericana de micologia: organo de la Asociacion Espanola de Especialistas en Micologia 2001;18:133–6. [28] Carvalho MTM, Castro AP, Baby C, Werner B, Neto JF, Queiroz-Telles F. Disseminated cutaneous sporotrichosis in a patient with AIDS: report of a case. Revista da Sociedade Brasileira de Medicina Tropical 2002;35(6):655–9. [29] Schubach TMP, Schubach AO, Okamoto T, Barros MBL, Figueiredo FB, Cuzzi T, et al. Canine sporotrichosis in Rio de Janeiro, Brazil: clinical presentation, laboratory diagnosis and therapeutic response in 44 cases (1998–2003). Medical Mycology 2006;44: 87–92. [30] Bernstein JA, Cook HE, Gill AF, Ryan KA, Sirninger J. Cytologic diagnosis of generalized cutaneous sporotrichosis in a hunting hound. Veterinary Clinical Pathology 2007;36(1):94–6. [31] Schubach TMP, Schubach AO, Okamoto T, Figueiredo FB, Pereira SA, Leme LRP, et al. Sporothrix schenckii isolation from blood clot of naturally infected cats. Brazilian Journal of Veterinary Research and Animal Science 2004;41(6):404–8. [32] Kong X, Xiao T, Lin J, Wang Y, Chen HD. Relationships among genotypes, virulence and clinical forms of Sporothrix schenckii infection. Clinical Microbiology and Infection 2006;12(11): 1077–81. [33] Arrillaga-Moncrieff I, Capilla J, Mayayo E, Marimon R, Mariné M, Gené J, et al. Different virulence levels of the species of Sporothrix in a murine model. Clinical Microbiology and Infection 2009;15(7):651–5.
432
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[34] Uenotsuchi T, Takeuchi S, Matsuda T, Urabe K, Koga T, Uchi H, et al. Differential induction of Th1-prone immunity by human dendritic cells activated with Sporothrix schenckii of cutaneous and visceral origins to determine their different virulence. International Immunology 2006;18(12):1637–46. [35] Reis RS, Almeida-Paes R, Muniz MM, Tavares PM, Monteiro PC, Schubach TM, et al. Molecular characterisation of Sporothrix schenckii isolates from human and cats involved in the sporotrichosis
epidemic in Rio de Janeiro, Brazil. Memorias do Instituto Oswaldo Cruz 2009;104(5):769–74. [36] Galhardo MCG, De Oliveira RMZ, Valle ACF, Paes RA, Silvatavares PM, Monzon A, et al. Molecular epidemiology and antifungal susceptibility patterns of Sporothrix schenckii isolates from a cat-transmitted epidemic of sporotrichosis in Rio de Janeiro, Brazil. Medical Mycology 2008;46:141–51.
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Feline sporotrichosis: Histopathological profile of cutaneous lesions and their correlation with clinical presentation Luisa H.M. Miranda a,∗ , Fátima Conceic¸ão-Silva b , Leonardo P. Quintella c , Bianca P. Kuraiem a , Sandro A. Pereira a , Tânia M.P. Schubach a a Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos, Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Fundac¸ão Oswaldo Cruz (FIOCRUZ), Av. Brasil, 4365, Rio de Janeiro 21040-360, Brazil b Laboratório de Imunoparasitologia, Instituto Oswaldo Cruz, FIOCRUZ, Pavilhão Leônidas Deane, Av. Brasil, 4365, Rio de Janeiro 21040-360, Brazil c Servic¸o de Anatomia Patológica, IPEC, FIOCRUZ, Pavilhão Gaspar Viana, Av. Brasil, 4365, Rio de Janeiro 21040-360, Brazil
a r t i c l e
i n f o
Article history: Received 30 August 2012 Received in revised form 23 January 2013 Accepted 30 March 2013 Keywords: Cat Sporotrichosis Histopathology Skin Sporothrix schenckii
a b s t r a c t Cutaneous lesions of feline sporotrichosis show high fungal load and are associated with severe disease and elevated zoonotic potential. The present study describes the histopathology and fungal load of the lesions in different clinical presentations of feline sporotrichosis. Cats with sporotrichosis were separated into groups L1, L2 and L3 (lesions in one, two and three or more locations, respectively) and subjected to skin biopsies for histopathology. Eighty-six cats were included in the study. Lesions were suppurative granulomatous in 84 cases and poorly formed granulomas were predominant. The well-formed granulomas were associated with group L1. The high fungal load was predominant in group L3 and in poorly formed granuloma cases and did not occur in well-formed granulomas cases. The good general condition was associated with low fungal load. These findings suggest that the fungal load control in animals with more localized lesions and well-organized response is linked with the improvement in the outcome of infected cats. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Sporotrichosis is a mycosis that affects humans and many animals worldwide, and is caused by Sporothrix schenckii complex species [1]. Transmission occurs through a traumatic inoculation through the skin with soil, plants, and organic matter contaminated with the fungus [2]. Zoonotic transmission may also occur through a bite, scratch or contact with the exudate from infected lesions. Cats have a high zoonotic potential, since their skin lesions contain plenty of yeast [3]. Since 1998, sporotrichosis has appeared as an epidemic disease in Rio de Janeiro, Brazil, and by 2009, there have been 2200 human cases, 3244 feline cases and 120 canine
∗ Corresponding author. Tel.: +55 21 38659536; fax: +55 21 38659553. E-mail address: [email protected] (L.H.M. Miranda). 0147-9571/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cimid.2013.03.005
cases diagnosed by the Evandro Chagas Clinical Research Institute (IPEC) – FIOCRUZ [4]. Among the human cases reported up till 2004, 83% have reported contact with sporotrichosis infected cats, and of these, 56% resulted from a scratch or bite of the animal [5]. Zoonotic transmission appears to explain the implementation of the epidemic and its maintenance in an endemic form [6]. Several species of the S. schenckii complex have been isolated from human and feline lesions in Rio de Janeiro, Brazil and Sporothrix brasiliensis has been considered the most prevalent species in this current epidemic [4,7], with a prevalence of more than 90.0% of cases. S. schenckii has also been described in few cases (6.0%). Sporothrix mexicana and Sporothrix globosa were observed in a single case by each one [8]. Currently, the cat is the animal species most affected by sporotrichosis [9] and their cutaneous lesions are characterized by subcutaneous nodules which develop into
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ulceration. The clinical signs in feline sporotrichosis are variable, with subclinical infections, single lesions, and fatal disseminated systemic forms [3,10]. Experimental studies in mice suggest that the histopathologic lesions of sporotrichosis vary with the stage of disease, showing first the formation of abscesses and the participation of macrophages and lymphocytes, associated with a high fungal load. When the macrophages activate and begin to differentiate, thereby forming granulomas, the fungal load decreases, accompanied by the reduction of abscesses and increase in plasma cells [11–13]. The occurrence of severe sporotrichosis, including skin lesions without granulomas and rich in fungal elements in cats that are apparently immunocompetent [3,10] show the high susceptibility of these animals to infection by S. schenckii and emphasize the need to better understand the immune response against this fungus and its association with the fungal load and general condition of the animals. The acquired results could help in the design of new drugs and in the treatment and control of feline sporotrichosis, further reducing its transmission to humans and other cats. This present study aims to describe a histopathological profile of feline sporotrichosis lesions and to verify the differences in pathological expression and fungal load, according to clinical presentation, age and general condition of the animals.
of 0.4 cm, after sedation and local anesthesia. The biopsy specimens were fixed in 10% buffered formalin for histological examination at the Department of Pathology, IPEC – FIOCRUZ. The procedures performed on the cats and the use and storage of their biological samples, were approved by the Animal Ethics Committee (CEUA), FIOCRUZ. License number LW-28/10. 2.2. Histopathology
2. Material and methods
Sections (5 ) were stained with haematoxylin and eosin (HE) and Grocott silver stain for microscopic analysis. Histopathological analysis was performed in an optical microscope by a trained technician. The lesions were described considering the type of inflammation – granulomatous or nonspecific. In cases of granulomatous inflammation, the cell activation degree of the mononuclear phagocytic system in granulomas – macrophages or epithelioid cells – and organization of the granuloma were also verified. Table 1 is adapted from a study of Miranda et al. [15] and represents definitions of the histopathological variables used in this study. In the Grocott silver stain analysis, the cases in which yeast-like forms (YLF) were present were considered positive. The cases were considered negative when no YLF was observed after the analysis of 50 high power microscopic fields (400×).
2.1. Sampling
2.3. Statistical analysis
The study was cross-sectional and samples were obtained from outpatient routines at the Domestic Animal Dermatozoonosis Laboratory of Clinical Research (LAPCLIN/DERMZOO) IPEC – FIOCRUZ, during the period of July 2009–August 2011. The animals considered eligible were cats with sporotrichosis (confirmed by isolation and identification of Sporothrix sp. complex in culture medium), showing ulcerated skin lesions, with no previous antifungal or corticosteroid therapy. Cases were excluded when the biopsy sample was considered superficial (not including at least the reticular dermis) or not representative (not exhibiting inflammatory infiltration covering more than one third of the sample). The general condition of the animals was classified as good, fair or poor. Good general condition was considered with the absence of extra-cutaneous signs (0). The presence of signs such as dyspnea, conjunctivitis, weight loss, dehydration, pale mucous membranes and prostration, when mild, indicate fair general condition (1 or 2), and when accented, poor general condition (3 or 4). The cats were divided into three groups according to the distribution of skin lesions: L1, L2 and L3 [3]. In L1, cats with lesions in one location were included; in L2, cats with lesions in two non-contiguous locations were included, and in L3, cats with lesions in three or more non-contiguous locations were included. Samples were obtained from exudates of skin ulcers by means of sterile swabs (isolation and identification of fungi in culture medium) [14] and from fragments of the edge of the ulcerated lesion by means of biopsy, using a punch
Data were stored and analyzed with the Statistical Package for Social Sciences (SPSS) for Windows, version 16.0 (SPSS Inc.). For descriptive analysis, we calculated the frequencies of the variables. The different groups were compared regarding the clinical findings and histopathological analysis. The chi-square test of independence was used to determine a significant association between the variables studied. Fisher’s exact test was applied to the comparison of variables with only two categories. In all specified analyses, a p-value < 0.05 was considered as the indication of a statistically significant association. 3. Results 3.1. Sampling One hundred and nine cats, seventy-nine males and thirty females, were observed from July 2009 to August 2011 and eighty-six were included in the study. Seventeen cats were not included due to previous anti-fungal or corticosteroid therapy. One cat was excluded for no isolation of fungi and five because biopsy samples were considered non-representative or superficial. 3.2. Histopathological findings Microscopically, 84 (97.7%) showed suppurative granulomatous inflammation and two (2.3%) showed nonspecific inflammation.
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Table 1 Terms and definitions used in the histopathological classification of feline sporotrichosis. Histopathological term Granuloma/granulomatous inflammation
Categories
Definition Inflammatory infiltrates contain activated cells of the mononuclear phagocyte system (MPS) forming compact aggregates or interstitial cords
Nonspecific inflammation
Inflammatory infiltrates without granuloma
Suppurative granuloma
Granuloma with neutrophil infiltration
Fungal granuloma
Granulomas in which macrophages are filled with yeast-like forms throughout the length of the granuloma. Reaction lympho-plasmacytic scarce Mild to moderate
Intensity of neutrophils infiltration of a granuloma
Marked Mild to moderate
Intensity of lymphocytes infiltration of a granuloma
Marked Mild to moderate
Intensity of plasma cells infiltration of a granuloma
Marked
Activated cells of the MPS in granulomas
Macrophages Epithelioid cells
Granuloma organization
Well formed Poorly formed
Infiltration of neutrophils into the granuloma is scattered, in loose and small clusters Infiltration of neutrophils into the granuloma is dense and more cellular than that of mild to moderate intensity infiltrates Infiltration of lymphocytes into the granuloma is scattered, in loose and small clusters Infiltration of lymphocytes into the granuloma is dense and more cellular than that of mild to moderate intensity infiltrates Infiltration of plasma cells into the granuloma is scattered, in loose and small clusters Infiltration of plasma cells into the granuloma is dense and more cellular than that of mild to moderate intensity infiltrates Large polygonal cells with plump eccentric nuclei and pale eosinophilic, slightly granular, cytoplasm with distinct borders Large elongate cells with eccentric reniform nuclei and abundant pale eosinophilic cytoplasm with indistinct borders Well delineated with a predominance of epithelioid cells Poorly delineated and/or a predominance of macrophages
MPS: mononuclear phagocytic system.
3.2.1. Suppurative granulomatous inflammation (n = 84) Eighty-four cases were characterized by suppurative granulomatous inflammation: 12 (14.3%) from group L1, 15 (17.9%) from L2 and 57 (67.9%) from L3. Table 2 shows the frequencies of the clinic-epidemiological variables for the cases characterized by suppurative granulomatous inflammation. The infection was predominant in males from all three groups, with significant difference in the L3 group (pvalue = 0.006). Among the cats of L1 (n = 12), eight were in good general condition and none of the cats included in groups L1 and L2 had poor general condition. Table 3 shows the frequencies of histopathological findings from each group. The proportion of poorly formed granulomas (Fig. 1A) was different amongst the groups:
Table 2 Feline sporotrichosis: frequencies of the clinical-epidemiological variables in cases characterized by suppurative granulomatous inflammation. Variable (n)
Lesions characterized by suppurative granulomatous inflammation (N = 84) n (%)
General condition (84)
Good Fair Poor
41 (48.8) 27 (36.1) 16 (19.1)
Age (79)
>0 < 2 years ≥2 < 5 years ≥5 years
26 (32.9) 43 (54.4) 10 (12.7)
Gender (84)
Male Female
62 (73.8) 22 (26.2)
75.0% in group L1, 80.0% in L2 and 94.6% in L3 (pvalue = 0.048). Fungal granulomas were observed in 34 cases (41.7%): 8.3% of cases from group L1, 26.7% from L2 and 51.8% from L3 (p-value = 0.008). There was a significant difference (p-value = 0.004) regarding the fungal granuloma and the general condition: among the 41 cats in good general condition, nine (22.0%) had fungal granulomas. In cats with fair and poor general condition, this percentage was 55.6% and 62.5%, respectively. In cats over five years of age (n = 10), the presence of epithelioid cells and well formed granulomas (Fig. 1B) had a percentage of 50.0% and 42.9% respectively. In cats of less than two and of two to five years aged, the presence of epithelioid cells had percentage of 8.0% and 11.6%, respectively (p = 0.005) and the well formed granulomas had percentage of 4.0% and 9.3%, respectively. The intensity of neutrophils was marked (Fig. 1C) in 55 (65.5%) cases: 58.2% in cats in good general condition, 29.1% in cats with fair general condition and 12.7% in cats with poor general condition. Among the cats in good general condition (n = 41), 32 (80.0%) exhibited marked intensity of neutrophils (p-value = 0.022). Among the cases with marked intensity of lymphocytes (n = 30), 66.7% had good general condition, 26.6% had fair general condition and 6.7% had poor general condition (p-value = 0.021). Among the cases with mild to moderate lymphocyte (n = 54), 22.4% were less than two years old, 69.4% were two to five years old and 8.2% were over five years old (pvalue = 0.004).
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Table 3 Feline sporotrichosis: frequencies of each histopathological finding in cases characterized by suppurative granulomatous inflammation in three groups. Histopathological finding
Clinical presentation L1 (N = 12) n (%)
Total L2 (N = 15) n (%)
L3 (N = 57) n (%)
n (%)
Granuloma organization
Poorly formed Well formed
9 (75.0) 3 (25.0)
12 (80.0) 3 (20.0)
54 (94.7) 3 (5.3)
75 (89.3) 9 (10.7)
Predominant activated cells of the MPS in granulomas
Macrophage Epithelioid cells
9 (75.0) 3 (25.0)
11 (73.3) 4 (26.7)
51 (89.5) 6 (10.5)
71 (84.5) 13 (15.5)
Fungal granuloma
Present Absent
1 (8.3) 11 (91.7)
4 (26.7) 11 (73.3)
29 (50.9) 28 (49.1)
34 (40.5) 50 (59.5)
Neutrophils infiltrate intensity
Mild to moderate Marked
1 (8.3) 11 (91.7)
5 (33.3) 10 (66.7)
23 (40.4) 34 (59.6)
29 (34.5) 55 (65.5)
Lymphocytes infiltrate intensity
Mild to moderate Marked
6 (50.0) 6 (50.0)
8 (53.3) 7 (46.7)
40 (70.2) 17 (29.8)
54 (64.3) 30 (35.7)
Plasma cells infiltrate intensity
Absent Mild to moderate Marked
0 4 (33.3) 8 (66.7)
1 (6.7) 7 (46.7) 7 (46.7)
0 28 (49.1) 29 (50.9)
1 (1.2) 39 (46.4) 44 (52.4)
L1: cats with lesions in one location; L2: cats with lesions in two non-contiguous locations; L3: cats with lesions in three or more non-contiguous locations. MPS: mononuclear phagocytic system.
3.2.2. Nonspecific inflammation (n = 2) Two cases showed no granulomas on histopathological analysis: one from the L1 group and another from the L2. Both were male and in good condition. One of them was less than two years old and the other was over five. The nonspecific inflammation showed marked intensity of neutrophils in both cases. The lymphocytes infiltration showed marked intensity in one case and mild to
moderate in the other. Plasma cells showed mild to moderate intensity in both cases. 3.2.3. Grocott silver stain In cases characterized by suppurative granulomatous inflammation (n = 84), the Grocott silver stain revealed YLF in 79 cases (94.0%): 11 from group L1, 12 from L2 and 56 from L3 (p-value = 0.027).
Fig. 1. Feline sporotrichosis. Skin. (A) Fungal granuloma. Poorly formed suppurative granuloma, in which macrophages are filled with yeast-like forms. Hematoxyline-Eosine. (B) Well-formed suppurative granuloma. Hematoxyline-Eosine. (C) Marked intensity of neutrophils. Hematoxyline-Eosine. (D) High fungal load. Grocott silver stain.
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Table 4 Feline sporotrichosis: evaluation of fungal load in the histological sections by the technique of Grocott silver stain regarding the clinical and epidemiological findings. Variable (n)
Fungal load at GSS Absent
Up to 100 yeast-like forms/field*
Over 100 yeast-like forms/field*
General condition n (%)
Good (41) Fair (27) Poor (16)
4 (9.7) 1 (3.7) 0
17 (41.5) 9 (33.3) 3 (18.8)
20 (48.8) 17 (63.0) 13 (81.2)
Age n (%)
>0 < 2 years (26) ≥2 < 5 years (43) ≥5 years (10)
0 2 (4.6) 2 (20.0)
12 (46.2) 11 (25.6) 4 (40.0)
14 (53.8) 30 (69.8) 4 (40.0)
Gender n (%)
Male (62) Female (22)
4 (6.4) 1 (4.5)
20 (32.3) 9 (40.9)
38 (61.3) 12 (54.6)
GSS: Grocott silver stain. * Microscope field of 400×.
In 45 (53.6%) cases, more than 400 YLF/field of 400× (Fig. 1D) were observed. This fungal load was observed in 25.0% of cases from group L1, 40.0% from L2 and 63.2% from L3. Tables 4 and 5 show the values of fungal load regarding the clinical, epidemiological and pathological data. In well-formed granulomas, a fungal load of greater than 100 YLF/field of 400× was not observed, in poorly formed granulomas, this fungal load was seen in 66.7% of cases (p-value = 0.001). In cases where granulomas showed a predominance of epithelioid cells, the fungal load exceeding 100 YLF/field was not observed. In granulomas with predominance of macrophages, this load was observed in 72.5% (p-value < 0.001) of cases. Among the cases that showed mild to moderate intensity of neutrophils infiltrate (n = 29), four (14.3%) had fungal load up to 100 YLF/field and 24 (82.1%) load of more than 100 YLF/field. Among the cases showing marked intensity (n = 55), four (7.3%) showed no YLF, 24 (43.6%) had fungal load up to 100 YLF/field and 27 (49.1%) had load higher than 100 YLF/field (p-value = 0.006). In both cases characterized by nonspecific inflammation, the Grocott silver stain showed YLF. One showed
fungal load up to 100 YLF/field and in the other, over 100 YLF/field. 4. Discussion In this study, lesions in multiple locations were prevalent, although half of the cats (50.0%) were in good general condition. This observation was previously described by Schubach et al. [16] and Pereira et al. [9]. In this study, none of cats in groups L1 and L2 had poor general condition. However, the involvement of systemic sporotrichosis by cats without the presence of clinical signs is common [16]. In all groups, there was predominance of males, and the percentage difference in relation to females was significant in the L3 group. The cutaneous lesions of feline sporotrichosis in this study were predominantly suppurative granulomatous and with poorly formed granulomas. Other authors have also described lesions of feline sporotrichosis as suppurative granulomatous [17,18]. However, in a previous study conducted in Rio de Janeiro, the presence of granulomas was not frequent (12.2%) [3,19]. There were no significant differences regarding the presence of granuloma and the different clinical presentations. In addition to the low
Table 5 Feline sporotrichosis: evaluation of fungal load in the histological sections by the technique of Grocott silver stain regarding the histopathologic findings. Histological finding (n)
Fungal load at GSS Absent
Up to 100 yeast-like forms/field*
Over 100 yeast-like forms/field*
Granuloma organization n (%)
Well-formed (9) Poorly formed (75)
3 (33.3) 2 (2.7)
6 (66.7) 22 (29.3)
0 51 (68.0)
Predominant MPS cells n (%)
Epithelioid cells (13) Macrophages (71)
4 (30.8) 1 (1.4)
9 (69.2) 19 (26.8)
0 51 (71.8)
Neutrophils infiltrate intensity n (%)
Mild to moderate (29) Marked (55)
1 (3.4) 4 (7.3)
4 (13.8) 24 (43.6)
24 (82.8) 27 (49.1)
Lymphocytes infiltrate intensity n (%)
Mild to moderate (54) Marked (30)
3 (5.6) 2 (6.7)
16 (29.6) 12 (40.0)
35 (64.8) 16 (53.3)
Plasma cells infiltrate intensity n (%)
Absent (1) Mild to moderate (39) Marked (44)
0 3 (7.7) 2 (4.5)
1 (100.0) 11 (28.2) 16 (36.4)
0 25 (64.1) 26 (59.1)
GSS: Grocott silver stain. MPS: mononuclear phagocytic system. * Microscope field of 400×.
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frequency of granuloma, the percentage of cats in Group L1 (32.8%) was higher [3] compared to that described in this present study (14.3%). This suggests that the tendency to formulate the more organized response described regarding the animals of group L1 was probably not observed before. The granulomas of human and canine sporotrichosis lesions are also predominantly poorly formed [20,21]. However, in dogs and humans, poorly formed granulomas usually show a predominance of epithelioid cells, while the poorly formed granulomas of the feline sporotrichosis had a predominance of macrophages in almost all the lesions described. Additionally, the high frequency of fungal granulomas in feline sporotrichosis reveals the minimal lympho-plasmacytic reaction, normally present in lesions of human and canine sporotrichosis. Marques et al. [22] described similar findings for feline sporotrichosis: a granulomatous infiltration composed primarily of macrophages full of fungal structures, and minimal lympho-plasmacytic reaction In experimental studies of sporotrichosis lesions in mice, the influx of mononuclear phagocytic system cells, their activation and subsequent differentiation into epithelioid cells occur along with the reduction of fungal load and the increase of lymphocytes and plasma cells infiltrated [10,11,23]. Thus, the predominance of macrophages in granulomas in lesions of feline sporotrichosis and the lack of a lympho-plasmacytic reaction may be a reflection of the susceptibility of cats to the fungus and may be related to the high fungal load observed in most cases. The presence of well-formed granulomas and epithelioid cells granulomas in cats over five years old was significant. It was also observed that the intensity of lymphocytes was related to the cat’s age, with smaller proportions of mild to moderate intensity in cats over five years old. The presence of poorly formed granulomas was associated with group L3 and the marked intensity of lymphocytes was associated with good general condition. Together, these findings suggest that cats over five years may have become more resistant to disease than the cats of other age groups. Sporotrichosis lesions in cats generally exhibit a lot of yeast forms. In this study, the detection of fungi was obtained in 79 (94.0%) cases by means of the Grocott silver stain technique, and the highest percentage was observed in lesions of cats from the L3 group. In 52.3% of the cases, more than 400 YLF/field (400×) were identified. This percentage was also higher in cats from group L3, indicating an association between fungal load and the clinical presentation of the animal. The fungal load is directly associated with the intensity of neutrophils in experimental and human sporotrichosis. In human sporotrichosis, the marked intensity of neutrophils is associated with patients with more lesions and longer treatment [24]. However, in this present study, we observed an inverse relationship between intensity of neutrophils and fungal load, with a high proportion of cases presenting high fungal load along with a mild to moderate intensity of neutrophils. Similar observations were described by Hachisuka and Sasai [25] in an experimental study, where immunosuppressed mice exhibited lesions
rich in fungi and with a scarcity of neutrophils and lymphocytes. Kajiwara et al. [26] attributed neutrophils to the involvement in the response of the fungus in mice. The greater efficiency of fungus phagocytosis by neutrophils in these animals could justify a lower fungal load in lesions with infiltrates rich in neutrophils. These results may also show different stages of inflammation: in immunocompetent patients, an increased influx of inflammatory cells in response to a high fungal load. In immunosuppressed patients, the highest concentration of fungal elements may occur by longitudinal growth due to lack of response. Previous studies during the epidemic in Rio de Janeiro also described an inverse relationship between fungal load and granuloma formation in the lesions of feline sporotrichosis [3,19]. However, the sensitivity of the histological method for detection of fungi and the frequency of granuloma were lower than those found in this study. In humans and dogs, the occurrence of severe forms of the disease seems related to immunosuppressive factors such as AIDS [27–30]. In cats, there was no further association between severity of sporotrichosis or fungal load of cutaneous lesions and infection by feline immunodeficiency virus and/or by feline leukemia virus [3,8,31]. Some experimental studies show that the more severe clinical presentations may be associated with more virulent strains of the fungus [32,33]. More virulent strains of S. schenckii induce a weak inflammatory response, facilitating the growth of fungus in the lesion [34]. In the present study, the identification of species of S. schenckii complex was not performed. However, since S. brasiliensis has been pointed as the major prevalent species in Rio de Janeiro, we can suggest that the different conditions observed in the groups L1, L2 and L3 may be related to factors other than the infective species. In addition, it should be noted that in this zoonotic epidemic, the same isolates of the fungi circulate between humans and cats [35], with severe forms of sporotrichosis observed frequently in cats, but not in humans, in which are usually associated with immunosuppressive or concomitant disease factors. This suggests that, besides the virulence of the fungus and the species involved, other factors enable the occurrence of severe disease in cats, despite the absence of immunosuppressive factors and the infective species. Galhardo et al. [36] described a method of molecular fingerprinting to identify the genotypes of S. schenckii isolates recovered from different clinical forms of human sporotrichosis in the epidemic in Rio de Janeiro. According to this study, there was no correlation between the subtypes generated from the strains and the clinical forms. The authors suggest that the main factor determining these clinical forms is probably related to the immune response of the patient. The results suggest a relationship between a well organized immune response (well-formed granulomas and presence of epithelioid cells) and control of fungal load, as well as its association with the general condition of the animal and clinical presentation of the disease. However, further studies are necessary to state whether the different clinical presentations of feline sporotrichosis, fungal load and inflammatory profile are somehow related to infection
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by different species of S. schenckii complex or to different susceptibilities of cats. In addition, studies regarding the immune response of feline sporotrichosis and the disease course in cats could contribute to the better understanding of its pathogenesis and whether the different clinical presentations and inflammatory profiles could be determinant to the prognosis of the disease. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgments The authors thank Isabella D.F. Gremião, Marcos F.L. de Almeida, Maria D.P.E. Espinoza, Francisco C.C. Rodrigues, Antonio C. da Silva, Patricia F. Pereira, Luiz C. Ferreira, Rosani S. Reis, Rodrigo C. Menezes, Amanda A.B. Kitada, Érica G. Reis and Elaine W. Souza for technical support. This study was partially financed by the Programa de Apoio a Pesquisa Estratégica em Saúde (PAPES V), FIOCRUZ, by the Fundac¸ão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and by the Conselho Nacional de Desenvolvimento Científico Tecnológico (CNPq), Brazil. TMPS is a CNPq researcher. References [1] López-Romero E, Reyes-Montes Mdel R, Pérez-Torres A, RuizBaca E, Villagómez-Castro JC, et al. Sporothrix schenckii complex and sporotrichosis, an emerging health problem. Future Microbiol 2011;6(1):85–102. [2] Rippon J. Sporotrichosis. Journal of Veterinary Internal Medicine 1988;7:44–5. [3] Schubach TMP, Schubach AO, Okamoto T, Barros MB, Figueiredo FB, Cuzzi T, et al. Evaluation of an epidemic of sporotrichosis in cats: 347 cases (1998–2001). Journal of the American Veterinary Medical Association 2004;224:1623–9. [4] Barros MB, de Almeida Paes R, Schubach AO. Sporothrix schenckii and sporotrichosis. Clinical Microbiology Reviews 2011;24(4):633–54. [5] Schubach AO, Barros MBL, Wanke B. Epidemic sporotrichosis. Current Opinions in Infectious Diseases (Philadelphia, PA) 2008;21:129–33. [6] Barros MBL, Schubach TP, Coll JO, Gremião ID, Wanke B, Schubach A, et al. Esporotricose: a evoluc¸ão e os desafios de uma epidemia. Revista Panamericana de Salud Publica 2010;227(6):455–60. [7] Marimon R, Cano J, Gené J, Sutton DA, Kawasaki M, Guarro J. Sporothrix brasiliensis, S. globosa, and S. mexicana, three new Sporothrix species of clinical interest. Journal of Clinical Microbiology 2007;45(10):3198–206. [8] Oliveira MM, Almeida-Paes R, Muniz MM, Gutierrez-Galhardo MC, Zancopé-Oliveira RM. Phenotypic and molecular identification of Sporothrix isolates from an epidemic area of sporotrichosis in Brazil. Mycopathologia 2011;172(4):257–67. [9] Pereira SA, Passos SRL, Silva JN, Gremião IDF, Figueiredo FB, Teixeira JL, et al. Response to azolic antifungal agents for treating feline sporotrichosis. Veterinary Record 2010;166(10):290–4. [10] Schubach TMP, Schubach AO, Cuzzi-Maya T, Okamoto T, Reis RS, Monteiro PCF, et al. Pathology of sporotrichosis in 10 cats in Rio de Janeiro. Veterinary Record 2003;152:172–5. [11] Miyaji M, Nishimura K. Defensive role of granuloma against Sporothrix schenckii infection. Mycopathologia 1982;80:117–24. [12] Mohri S. Study in sporotrichosis – III. Histological and Immunohistochemical study in experimental cutaneous sporotrichosis in man. Yokohama Medical Bulletin 1987;38(1–2):37–48. [13] Hiruma M, Yamaji K, Shimizu H, Ohata H, Kukita A. Ultrastructural study of tissue reaction of mice against Sporothrix schenckii infection. Archives for Dermatological Research 1988;280(Suppl.):94–100.
431
[14] Kwon-Chung K, Bennett J. Sporotrichosis. In: Kwon-Chung K, Bennett J, editors. Medical Mycology – The Pathogenic Fungi and the Pathogenic Actinomycetes. Philadelphia: Lea & Febiger; 1992. p. 707–29. [15] Miranda LHM, Quintella LP, Santos IB, Oliveira RVC, Menezes RC, Figueiredo FB, et al. Comparative histopathological study of sporotrichosis and American tegumentary leishmaniosis in dogs from Rio de Janeiro. Journal of Comparative Pathology 2010;143(1): 1–7. [16] Schubach TMP, Schubach AO, Okamoto T, Pellon IV, Monteiro PCF, Reis RS, et al. Haematogenous spread of Sporothrix schenckii in cats with naturally acquired sporotrichosis. Journal of Small Animal Practice 2003;44:395–8. [17] Dunstan RW, Langham RF, Reinman KA, Wakenell OS. Feline sporotrichosis: a report of five cases with transmission to humans. Journal of the American Academy of Dermatology 1986;15: 37–45. [18] Gonzalez Cabo JF, Heras Guillamon M, Latre Cequiel MV, Garcia de Jalon Ciercoles JA. Feline sporotrichosis: a case report. Mycopathologia 1989;108:149–54. [19] Schubach TMP, Schubach AO, Reis RS, Cuzzi-Maya T, Blanco TCM, Monteiro DF, et al. Sporothrix schenckii isolated from domestic cats with and without sporotrichosis in Rio de Janeiro, Brazil. Mycopathologia 2002;153:83–6. [20] Miranda LHM, Quintella LP, Santos IB, Menezes RC, Figueiredo FB, Gremião IDF, et al. Histopathology of canine sporotrichosis: a morphological study of 86 cases from Rio de Janeiro (2001–2007). Mycopathologia 2009;168(2):79–87. [21] Quintella LP, Passos SR, do Vale AC, Galhardo MC, Barros MB, Cuzzi T, et al. Histopathology of cutaneous sporotrichosis in Rio de Janeiro: a series of 119 consecutive cases. Journal of Cutaneous Pathology 2011;38(1):25–32. [22] Marques SA, Franco SRVS, Camargo RMP, Dias LDF, Haddad Jr V, Fabris VE. Esporotricose do gato doméstico (Felis catus): transmissão humana. Revista do Instituto de Medicina Tropical de São Paulo 1993;35(4):327–30. [23] Peng-Cheng L, Yoshiike T, Yaguchi H, Ogawa H. Histopathological studies of Sporothrix schenckii-inoculated mice – possible functions of polymorphonuclear leukocytes in normal and immunocompromised (congenitally athymic nude) mice. Mycopathologia 1993;122: 89–93. [24] Morgado FN, Schubach AO, Barros MBL, Conceic¸ão-Silva F. The in situ inflamatory profile of lymphocutaneus and fixed forms of human sporotrichosis. Medical Mycology 2011;49(6):612–20. [25] Hachisuka H, Sasai Y. Development of experimental sporotrichosis in normal and modified animals. Mycopathologia 1981;76: 79–82. [26] Kajiwara H, Saito M, Ohga S, Uenotsuchi T, Yoshida S. Impaired host defense against Sporothrix schenckii in mice with chronic granulomatous disease. Infection and Immunity 2004;72(9): 5073–9. [27] Rocha MM, Dassin T, Lira R, Lima EL, Severo LC, Londero AT. Sporotrichosis in a patient with AIDS: report of a case and review. Revista iberoamericana de micologia: organo de la Asociacion Espanola de Especialistas en Micologia 2001;18:133–6. [28] Carvalho MTM, Castro AP, Baby C, Werner B, Neto JF, Queiroz-Telles F. Disseminated cutaneous sporotrichosis in a patient with AIDS: report of a case. Revista da Sociedade Brasileira de Medicina Tropical 2002;35(6):655–9. [29] Schubach TMP, Schubach AO, Okamoto T, Barros MBL, Figueiredo FB, Cuzzi T, et al. Canine sporotrichosis in Rio de Janeiro, Brazil: clinical presentation, laboratory diagnosis and therapeutic response in 44 cases (1998–2003). Medical Mycology 2006;44: 87–92. [30] Bernstein JA, Cook HE, Gill AF, Ryan KA, Sirninger J. Cytologic diagnosis of generalized cutaneous sporotrichosis in a hunting hound. Veterinary Clinical Pathology 2007;36(1):94–6. [31] Schubach TMP, Schubach AO, Okamoto T, Figueiredo FB, Pereira SA, Leme LRP, et al. Sporothrix schenckii isolation from blood clot of naturally infected cats. Brazilian Journal of Veterinary Research and Animal Science 2004;41(6):404–8. [32] Kong X, Xiao T, Lin J, Wang Y, Chen HD. Relationships among genotypes, virulence and clinical forms of Sporothrix schenckii infection. Clinical Microbiology and Infection 2006;12(11): 1077–81. [33] Arrillaga-Moncrieff I, Capilla J, Mayayo E, Marimon R, Mariné M, Gené J, et al. Different virulence levels of the species of Sporothrix in a murine model. Clinical Microbiology and Infection 2009;15(7):651–5.
432
L.H.M. Miranda et al. / Comparative Immunology, Microbiology and Infectious Diseases 36 (2013) 425–432
[34] Uenotsuchi T, Takeuchi S, Matsuda T, Urabe K, Koga T, Uchi H, et al. Differential induction of Th1-prone immunity by human dendritic cells activated with Sporothrix schenckii of cutaneous and visceral origins to determine their different virulence. International Immunology 2006;18(12):1637–46. [35] Reis RS, Almeida-Paes R, Muniz MM, Tavares PM, Monteiro PC, Schubach TM, et al. Molecular characterisation of Sporothrix schenckii isolates from human and cats involved in the sporotrichosis
epidemic in Rio de Janeiro, Brazil. Memorias do Instituto Oswaldo Cruz 2009;104(5):769–74. [36] Galhardo MCG, De Oliveira RMZ, Valle ACF, Paes RA, Silvatavares PM, Monzon A, et al. Molecular epidemiology and antifungal susceptibility patterns of Sporothrix schenckii isolates from a cat-transmitted epidemic of sporotrichosis in Rio de Janeiro, Brazil. Medical Mycology 2008;46:141–51.