Evaluation of immunohistochemistry for the diagnosis of sporotrichosis in dogs.
ABSTRACT The aim of this study was to apply immunohistochemistry (IHC) for the diagnosis of canine sporotrichosis and to compare this method with the Grocott's silver stain (GSS) and periodic acid Schiff (PAS) techniques. Eighty-seven dogs with sporotrichosis (group 1) and 35 with American tegumentary leishmaniosis (ATL) (group 2) were studied. The fungus was detected in group 1 by GSS, PAS and IHC. IHC was also applied to group 2 to evaluate the occurrence of cross-reactions. PAS, GSS and IHC detected yeast cells in 19.5%, 43.7% and 65.5% of the group 1 cases, respectively. The detection of intracellular antigens of Sporothrix schenckii by IHC increased the sensitivity of the histological diagnosis to 80.5%. No positive reaction was observed in ATL lesions. The results suggest that IHC may be indicated for the diagnosis of sporotrichosis because of its higher diagnostic sensitivity.
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ABSTRACT: Miltefosine (MIL), originally developed for use in cancer chemotherapy, has been shown to have important antifungal activity against several pathogenic fungi. Our aim in this study was to determine the in vitro activity of MIL against the dimorphic fungi Histoplasma capsulatum and Sporothrix spp. This was done using the broth microdilution method. MIL had an in vitro inhibitory effect against all strains of H. capsulatum var. capsulatum and Sporothrix spp. analyzed. The minimal inhibitory concentrations (MIC) varied from 0.25 μg/ml to 2 μg/ml for H. capsulatum var. capsulatum in the filamentous phase and from 0.125 μg/ml to 1 μg/ml in the yeast phase. The MIC interval for Sporothrix spp. in the filamentous phase was 0.25-2 μg/ml. The minimal fungicidal concentrations (MFCs) were ≤4 μg/ml for isolates of both analyzed species. This study demonstrates that MIL has an antifungal effect in vitro against two potentially pathogenic fungi and that more studies should be performed in order to evaluate its applicability in vivo.Medical mycology: official publication of the International Society for Human and Animal Mycology 03/2014; · 2.26 Impact Factor
Evaluation of immunohistochemistry for the diagnosis of sporotrichosis in dogs
Luisa H.M. Mirandaa,⇑, Leonardo P. Quintellab, Rodrigo C. Menezesa, Isabele B. dos Santosa,
Raquel V.C. Oliveirac, Fabiano B. Figueiredoa, Leila M. Lopes-Bezerrad, Tânia M.P. Schubacha
aLaboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos (LAPCLIN-DERMZOO), Instituto de Pesquisa Clínica Evandro Chagas,
Fundação Oswaldo Cruz (IPEC, FIOCRUZ), Av. Brasil, 436, Manguinhos, 21040-360 Rio de Janeiro, RJ, Brazil
bServiço de Anatomia Patológica (SEAP), IPEC, FIOCRUZ, Av. Brasil, 4365, Manguinhos, 21040-360 Rio de Janeiro, RJ, Brazil
cLaboratório de Epidemiologia Clínica, IPEC, FIOCRUZ, Av. Brasil, 4365, Manguinhos, 21040-360 Rio de Janeiro, RJ, Brazil
dLaboratório de Micologia Celular e Proteômica, IBRAG, Universidade do Estado do Rio de Janeiro (UERJ), R. São Francisco Xavier, 524, Maracanã, 20550-900 Rio de Janeiro, RJ, Brazil
a r t i c l ei n f o
Accepted 3 December 2010
a b s t r a c t
The aim of this study was to apply immunohistochemistry (IHC) for the diagnosis of canine sporotrichosis
and to compare this method with the Grocott’s silver stain (GSS) and periodic acid Schiff (PAS) tech-
niques. Eighty-seven dogs with sporotrichosis (group 1) and 35 with American tegumentary leishmani-
osis (ATL) (group 2) were studied. The fungus was detected in group 1 by GSS, PAS and IHC. IHC was also
applied to group 2 to evaluate the occurrence of cross-reactions. PAS, GSS and IHC detected yeast cells in
19.5%, 43.7% and 65.5% of the group 1 cases, respectively. The detection of intracellular antigens of
Sporothrix schenckii by IHC increased the sensitivity of the histological diagnosis to 80.5%. No positive
reaction was observed in ATL lesions. The results suggest that IHC may be indicated for the diagnosis
of sporotrichosis because of its higher diagnostic sensitivity.
? 2010 Elsevier Ltd. All rights reserved.
Sporotrichosis is a subcutaneous mycosis caused by the dimor-
phic fungus Sporothrix schenckii, which infects humans and several
animal species (Schell, 1998). Canine sporotrichosis is considered
to be rare and presents a low zoonotic potential (Santos et al.,
2007) but has reached epidemic proportions in Rio de Janeiro since
1998, affecting humans, dogs and cats (Schubach et al., 2008). The
increasing number of cases emphasizes the importance of fast and
efficient diagnostic methods for the disease. American tegumen-
tary leishmaniosis (ATL) has become the main differential diagno-
sis for sporotrichosis in humans and dogs in Rio de Janeiro (Barros
et al., 2005; Santos et al., 2007), and, in addition to their clinical
similarity, the endemic areas for the diseases overlap and cross-
reactions are observed in serological tests.
The reference method for the diagnosis of sporotrichosis is the
isolation and identification of the fungus in culture (Kwon-Chung
and Bennett, 1992). However, no fungal growth may be observed,
generally because of inadequate transport of the material or con-
tamination with saprophytic microorganisms (Moore and Acker-
man, 1946; Schwarz, 1982). Additionally, the fungus may present
slow growth, requiring maintenance of the culture for a long peri-
od (Schwarz, 1982).
Identification of S. schenckii in paraffin blocks is frequently the
only method available, particularly when fungal infection was
not suspected at the time of biopsy (Moskowitz et al., 1986;
Werner and Werner, 1994). Histochemical techniques for the
detection of fungal structures in histological sections, such as
Grocott’s silver stain (GSS) and periodic acid Schiff (PAS), are not
specific and present low sensitivity for the diagnosis of sporotri-
chosis in humans and dogs because of the scarcity of S. schenckii
yeast cells in skin lesions (Marques et al., 1992; Barros et al.,
2004, 2005; Schubach et al., 2006; Santos et al., 2007).
IHC has shown improved sensitivity in the diagnosis of human
sporotrichosis (Marques et al., 1992; Rodriguez and Sarmiento,
1998), but its application to the diagnosis of the disease in dogs
has not been standardized. The aim of the present study was to ap-
ply IHC for the diagnosis of canine sporotrichosis and to compare
this method with the GSS and PAS techniques.
Materials and methods
Active skin lesions obtained from dogs seen at the Evandro Chagas Clinical Re-
search Institute, Oswaldo Cruz Foundation, Instituto de Pesquisa Clínica Evandro
Chagas (IPEC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, between 2001
and 2008 were studied. The study was approved by the Ethics Committee on the
Use of Animals, FIOCRUZ (protocol P.00060-00).
The samples were embedded in paraffin blocks and divided into two groups:
group 1 consisted of skin lesions from 87 dogs with sporotrichosis confirmed by iso-
lation of S. schenckii in culture; group 2 consisted of skin lesions from 35 dogs with
1090-0233/$ - see front matter ? 2010 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Tel.: +55 21 3865 9536.
E-mail address: email@example.com (L.H.M. Miranda).
The Veterinary Journal 190 (2011) 408–411
Contents lists available at ScienceDirect
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ATL confirmed by isolation and characterization of Leishmania (Viannia) braziliensis.
The isolation of the agent in culture is considered the gold standard for the diagno-
sis of both diseases.
The GSS and PAS techniques were applied to group 1 for the detection of fungal
elements. The cases were qualitatively classified as positive or negative. Cases were
considered positive when yeast forms consistent with S. schenckii were found, and
negative when no yeast cell was observed after analyzing a range of 50 microscopic
fields of 400? magnification.
IHC using rabbit anti-S. schenckii polyclonal serum
The polyclonal serum was obtained according to protocol by Lopes-Alves et al.
(1994). Paraffin blocks were cut and mounted on silanated slides. After deparaffin-
ization and rehydration, endogenous peroxidase activity was blocked with 30%
hydrogen peroxide in 40 mL/100 mL (v/v) methanol solution. Nonspecific reactions
were inhibited with normal swine serum (Novocastra) in 1.5% bovine serum albu-
min (BSA) (1:20 dilution), followed by incubation in a solution of 0.1 g/mL milk
powder in 3.0% BSA.
Sections were then incubated in a moist chamber overnight at 4 ?C with anti-S.
schenckii serum in 1.5% BSA (1:4000 dilution); next they were washed with Tris-
buffered saline and incubated with universal biotinylated secondary antibody and
the streptavidin–biotin–peroxidase complex (DakoCytomation). The reaction was
developed using diaminobenzidine (DakoCytomation) as chromogen. The histolog-
ical sections were counterstained with Mayer’s hematoxylin and dehydrated.
The case used as positive control was confirmed by isolation of S. schenckii in
culture and was known to contain yeast cells on histopathological analysis. The
same case was also used as negative control by incubating with normal rabbit
serum (DakoCytomation) and BSA to control for nonspecific reactions and for
endogenous biotin and peroxidase activity, respectively. The analysis was con-
ducted as described for the GSS and PAS techniques. The pathologist was not
blinded to the diagnosis of the cases at the time of the analysis.
Data were stored and analyzed with SPSS for Windows, version 16.0. For the
analysis, the cases were classified as positive or negative based on the presence
of yeast forms consistent with S. schenckii. All cases included were previously con-
firmed as sporotrichosis by isolation of S. schenckii in culture. The sensitivity of the
methods (GSS, PAS and IHC) was calculated by comparing them to the culture re-
sults, which were considered the gold standard. The results were compared using
the McNemar test. P < 0.05 was considered significant.
The sensitivity of GSS staining (43.7%) was higher than that of
PAS staining (19.5%) (P < 0.001) for lesions of the 87 group 1 dogs
(sporotrichosis). GSS was positive in all PAS-positive cases. Yeast
cells were detected by IHC in 65.5% of the lesions (Fig. 1). Of these,
47.4% were negative by GSS. Eight of the 30 IHC-negative cases
were positive by GSS. The sensitivity of IHC was higher than that
of GSS (P = 0.002) and PAS (P < 0.001).
Intracellular antigens were detected by IHC in 27.6% of the cases
(Fig. 2). Among the cases in which yeast cells were not detected by
GSS or IHC (n = 22), five presented intracellular antigens by IHC.
Considering the detection of yeast cells and intracellular antigens,
the combined application of IHC and GSS increased the sensitivity
(80.5%) when compared to either technique alone.
The sensitivities of the single and combined methods are shown
in Table 1 and the sensitivities of the different techniques are
compared in Table 2. No positive reaction was observed for the
Fig. 1. Skin of a dog with sporotrichosis. S. schenckii yeast cells inside a suppurative
granuloma. Immunohistochemistry (anti-S. schenckii anti-serum). Bar = 10 lm.
Fig. 2. Skin of a dog with sporotrichosis. Intracellular antigens of S. schenckii inside
granulomas. Immunohistochemistry (anti-S. schenckii anti-serum). Bar = 10 lm.
Sensitivity of the different techniques, alone or in
combination, for the diagnosis of canine sporotrichosis.
TechniqueSensitivity (n = 87)
IHC (yeast + Ag)
IHC (yeast) + GSS
IHC (yeast + Ag) + GSS
PAS, periodic acid Schiff; GSS, Grocott’s silver stain;
IHC, immunohistochemistry; Ag, antigens.
Comparison of the sensitivity of the different techniques, alone or in combination, for
the diagnosis of canine sporotrichosis.
Technique(s)McNemar test (P?)
GSS ? PAS
GSS ? IHC (yeast)
PAS ? IHC (yeast)
GSS ? IHC (yeast + Ag)
PAS ? IHC (yeast + Ag)
IHC (yeast) ? IHC(yeast + Ag)
GSS ? IHC (yeast) + GSS
IHC (yeast) ? IHC (yeast) + GSS
GSS ? IHC (yeast + Ag) + GSS
IHC (yeast) ? IHC (yeast + Ag) + GSS
PAS, periodic acid Schiff; GSS, Grocott’s silver stain; IHC, immunohistochemistry;
Ag, antigens;?, P < 0.05 was considered statistically significant.
L.H.M. Miranda et al./The Veterinary Journal 190 (2011) 408–411
negative controls (Fig. 3). Cross-reactions with Leishmania spp.
amastigote forms or their antigens were not observed in lesions
of the 35 group 2 dogs (ATL).
Among the histological techniques studied, GSS showed higher
sensitivity than PAS for the diagnosis of canine sporotrichosis. Sim-
ilar results have been reported by Marques et al. (1992) and
Moskowitz et al. (1986) for humans. However, artifacts are fre-
quent in silver stains and might be mistaken for fungal structures,
particularly when the latter are scarce in the tissue (Moskowitz
et al., 1986).
In the present study, IHC showed higher sensitivity than GSS
and PAS in the detection of yeast cells in canine sporotrichosis le-
sions, a finding already described for human sporotrichosis by
Marques et al. (1992). Moskowitz et al. (1986) did not observe dif-
ferences in sensitivity between silver staining and IHC. Even when
scarce, S. schenckii yeast cells are easily detectable by IHC because
they acquire a brown color that contrasts on the blue background
of the tissue (Moskowitz et al., 1986; Marques et al., 1992).
The combination of GSS and IHC significantly increased sensi-
tivity when compared to IHC alone. Marques et al. (1992) also
found improved sensitivity in the diagnosis of human sporotricho-
sis when these two techniques were combined. However, despite
the improved sensitivity, it should be stated that the GSS stain is
not a specific method and is not indicated for differentiate yeast
cells of S. schenckii from other fungal species. Regarding the IHC
method, the results may vary according to the source of the anti-
body used, since this is not a commercially available antibody.
The IHC technique used in this study permitted the staining of
S. schenckii yeast cells and intracellular antigens. The labeling of
intracellular antigens by IHC has been described for leishmaniosis
lesions and is referred to as ‘cellular pattern’ (Schubach et al.,
2001). The detection of a sporotrichosis ‘cellular pattern’ in lesions
with a suspicion of sporotrichosis might be useful in cases in which
intact yeasts are not observed (Marques et al., 1992). However, the
diagnostic value of this finding requires further evaluation.
The sporotrichosis ‘cellular pattern’ frequently observed in the
present study increased the sensitivity of IHC to 73.6%. Thus, con-
sidering the detection of yeast cells and of the ‘cellular pattern’, the
combination of IHC and GSS increased the sensitivity of histologi-
cal diagnosis to 80.5%. This improved sensitivity was statistically
significant when compared to the results obtained for the methods
alone or in combination. Marques et al. (1992) also obtained
improved sensitivity of detection of the ‘cellular pattern’ and
S. schenckii yeast cells by IHC and emphasized the possible
contribution of these findings to the study of the pathogenesis of
sporotrichosis. Rodriguez and Sarmiento (1998) observed a sporo-
trichosis ‘cellular pattern’ in human sporotrichosis lesions and re-
lated this finding to fungal degradation by phagocytosis.
The relevance of the negative controls should be emphasized.
The ‘cellular pattern’ was considered to be specific in this study
due to its absence in the negative controls. Additionally, anti-S.
schenckii serum did not react with Leishmania spp. in canine ATL le-
sions. Previous studies in humans have reported cross-reactions
between S. schenckii and Leishmania spp. when anti-Leishmania ser-
um was used for IHC (Schubach et al., 2001; Quintella et al., 2009).
In dogs, the differential diagnosis between sporotrichosis and ATL
is usually difficult, because the diseases share clinical and epidemi-
ological features and present cross-reactions in serological tests
(Santos et al., 2007). Based on this evidence, the application of a
method which accurately differentiates the diseases is necessary.
In the present study, the IHC method, using the anti-S. schenckii
serum, showed specificity when applied on canine ATL lesions used
as negative controls, since the amastigotes and antigens of Leish-
mania were not visualized. Therefore, the technique described
might be a useful alternative or complementary technique for
the differential diagnosis between these diseases.
Since yeast cells are rare in most cases, the investigation of se-
rial sections obtained from the same fragment may increase the
chance of detection of the agent. In the present study, two sections
per case were analyzed by each technique, providing a higher sen-
sitivity when compared to other studies (Moraes and Miranda,
1964; Barros et al., 2005; Schubach et al. 2006; Santos et al.,
2007). Other authors reported a larger number of positive results
when multiple sections were analyzed (Moraes and Miranda,
1964; Itoh et al., 1986). According to Marques et al. (1992), the
deeper the skin fragments, the higher the possibility of detecting
the agent. However, skin biopsy fragments are usually small and
may not be sufficient to obtain serial sections.
GSS staining should be used if histopathological aspects consis-
tent with sporotrichosis are observed. IHC is a complementary
method that can be applied, if available, to improve the sensitivity
of the histological diagnosis.
Conflict of interest statement
None of the authors of this paper has a financial or personal
relationship with other people or organizations that could inappro-
priately influence or bias the content of the paper.
The authors thank Isabella D.F. Gremião, Sandro A. Pereira,
Thais Okamoto, Marcos F.L. de Almeida, Maria D.P.E. Espinoza,
Francisco C.C. Rodrigues, Antonio Correia, Antonio C. da Silva,
Rosani S. Reis, Paulo C.F. Monteiro, Maria de Fátima Madeira and
Rodrigo Méxas for technical support. This study was partially fi-
nanced by the Programa de Apoio a Pesquisa Estratégica em Saúde
(PAPES IV), FIOCRUZ, by the Fundação 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.
Fig. 3. Skin of a dog with sporotrichosis. Intracellular S. schenckii yeast cells
(arrowheads) without positive reaction. Immunohistochemistry negative control
(normal rabbit serum). Bar = 10 lm.
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