| Abstract|| |
Background: Diagnosis of chromoblastomycosis is frequently missed for many reasons: (1) rarity of the lesion (2) requirement of careful search for diagnostic 'sclerotic' bodies which are often sparse in clinical material such as tissue and exudates (3) often they elicit tissue reactions such as verrucous lesion and micro abscesses, misleading the diagnosis (4) lack of 'clinical suspicion'. Aims: (1) To compare the feasibility of 'unstained', 'stained' and 'de stained' sections in identification of the diagnostic ' sclerotic' bodies (2) To study histopathological features of chromoblastomycosis, and (3) To highlight the importance of clinical suspicion in the diagnosis of chromoblastomycosis. Design : This is a retrospective study conducted on six clinically unsuspected, histopathologically diagnosed cases of chromoblastomycosis. Results: Most common clinical presentation was verrucous lesion over extremities affecting adult men engaged in outdoor works. Sclerotic bodies and their characteristic features were easily identified in both 'unstained' and 'de stained' sections. Special stains such as Fontana Masson and Gomori's methenamine silver nitrate are useful for demonstrating hyphal forms in keratinized layers and for illustration purposes. Conclusion: Both 'unstained' and 'de stained' sections can be used for rapid screening of sclerotic bodies. 'De stained' sections can be used as a suitable substitute for special stains for confirmation and for retrospective review of all verrucous lesions to diagnose the missed lesion. Clinical suspicion is very important in the diagnosis of chromoblastomycosis as it helps pathologist to screen for diagnostic sclerotic bodies.
Keywords: Chromoblastomycosis, de stained sections, Fonsecea pedrosoi, phaeohyphomycosis, pseudoepitheliomatous hyperplasia, sclerotic bodies, transepithelial migration, unstained sections
|How to cite this article:|
Chavan SS, Kulkarni M H, Makannavar J H. 'Unstained' and 'de stained' sections in the diagnosis of chromoblastomycosis: A clinico-pathological study. Indian J Pathol Microbiol 2010;53:666-71
|How to cite this URL:|
Chavan SS, Kulkarni M H, Makannavar J H. 'Unstained' and 'de stained' sections in the diagnosis of chromoblastomycosis: A clinico-pathological study. Indian J Pathol Microbiol [serial online] 2010 [cited 2013 May 21];53:666-71. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/666/72021
| Introduction|| |
Phaeohyphomycosis are a heterogenous group of fungal lesions caused by dematiaceous fungi (pigmented fungi). Natural pigmentation is a chararacteristic property of these fungi. This property of natural pigmentation can be utilized for their own identification in clinical materials such as exudates and tissue samples. Some of these agents such as Fonsecea pedrosoi produced sclerotic bodies characteristic of chromoblastomycosis. These diagnostic sclerotic bodies are often small, sparse, seen in the background of an otherwise nonspecific inflammatory reaction and are frequently missed histologically. Hence it is challenging for pathologists to identify the lesion, who frequently receive the request with 'no clinical suspicion' of the lesion, which otherwise would have helped them to search for diagnostic 'sclerotic bodies'. This study discusses six clinically unsuspected and histologically confirmed cases of chromoblastomycosis, their clinical features, utility of 'unstained', 'de stained' and stained sections in its diagnosis, histopathology and the cytodiagnosis.
| Materials and Methods|| |
The paraffin blocks of six histopathologically diagnosed cases of chromoblastomycosis were retrieved and their clinical details, including the status of mycological culture studies, recorded. The unstained, stained (Hematoxylin and Eosin, Gomori's Methenamine silver nitrate and Fontana Masson) and de stained sections were examined for diagnostic sclerotic bodies. The ease with which the sclerotic bodies were identified (in terms of time taken) in hematoxylin and eosin stained sections, unstained sections as well as after de staining the H and E stained sections was observed and compared. One per cent acid alcohol is used for de staining. HandE stained sections (as well as smears) were kept in acid alcohol for 5- 10 minutes or more until complete decolorization.
| Results|| |
A total of six cases of chromoblastomycosis with varied clinical presentation were observed [Table 1]. Most occurred in men engaged in outdoor activities and one in an adolescent boy, with male to female ratio being 5:1. The most common site involved was lower extremity (83.33%) and the least was upper extremity. Most common presentation was cauliflower like warty growth [66.66%, in 4 cases] [Figure 1]a followed by non-healing ulcer and polypoid mass one in each of the remaining two cases. Characteristic tiny dark spots were seen in all six cases, especially on tips of papillae in case of cauliflower growths [Figure 1]b and on indurated area in non-healing ulcer. All six patients were apparently healthy with no significant associated illnesses or history or findings suggestive of immunodeficiency. Maximum duration of lesion was 16 years followed by 13 years, 11 years, 41/2 years, three years and least was six months. Routine investigations are within normal limits in all six cases with hemoglobin level in them ranged from 10.1g/dl to 14.1g/dl. The clinical diagnosis was lupus vulgaris, warty lesion, epithelioma, epithelioma, papilloma and epithelioma in six cases respectively. Mycological culture studies were not done for all six cases. Histologically, [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6] most consistent finding was microabscesses [Figure 2]b with mixed inflammatory cells, observed in all six cases and other prominent findings include pseudo-epitheliomatous hyperplasia [Figure 2]b and giant cell granulomatous response [Figure 2]a observed in 5 cases (83.33%) [Table 2]. Diagnostic sclerotic bodies were observed in all six cases and occurred in singles as well as in groups; chains of two or four [Figure 5]b, measured 5- 8μm with characteristic 'planate division' [Figure 5]c, some with triradiate [Figure 5]d or quadriradiate division [Figure 6]d and some showed crescent shape [Figure 5]c. The sclerotic bodies were seen lying freely in microabscesses [Figure 2]b, in giant cells [Figure 2]a and [Figure 5]a and also in keratinized layers [Figure 4]a-d.
|Figure 1 :Gross features of chromoblastomycosis: (a) cauliflower growth over dorsum of right foot (Case 3) with satellite lesions (arrow head). (b) cauliflower growth (Case 4) with characteristic black spots (arrow head)|
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|Figure 2 :Sclerotic bodies (arrow head and inset) in giant cell (a), micro abscess (b), eliciting pseudoepitheliomatous hyperplasia (b) (long arrow) (H and E, ×10), Inset: (H and E, ×40)|
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|Figure 3 :Cytology smear showing sclerotic bodies with characteristic equatorial division (arrow heads) in a background of erythrocytes and lymphocytes (arrow) (H and E, ×40). Inset: showing characteristic equatorial division|
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|Figure 4 :'Transepithelial migration' of sclerotic' bodies and unusual hyphal forms (arrow heads) in keratinized layers. (a) (H and E, ×10) (b)(H and E, ×40) (c) and (d) (Gomori's methenamine silver nitrate stain, ×10 and ×100 respectively)|
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|Figure 5 :Sclerotic bodies in unstained sections in groups with planate division (arrow head) (b), showing crescent shape(arrow head) (c) copper penny appearance(d) and triradiate division(arrow head) (e); in 'de stained' section in a giant cell(a). (f) An artifact in unstained section (arrow) (×40)|
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|Figure 6 :Fontana Masson stained sections showing sclerotic body with (arrow heads) (a) in a giant cell (FM, ×40) (b) 'copper penny' appearance (FM, ×40) (c) in micro abscess (FM, ×10), and (d) quadriradiate division (FM, ×40)|
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Diagnostic sclerotic bodies were easily identified in all the six cases and their morphology is very well characterized in both 'unstained' [Figure 5]b-e and 'de stained' sections [Figure 5]a in Fontana Masson [Figures 6]a-d and Gomori's methenamine silver stained sections [Figure 4]c and d, while they were identified with much effort and clues (such as giant cell granulomas) in H and E stained sections [Table 2]. In both stained and unstained sections sclerotic bodies resembled 'copper penny' [Figure 2]a, [Figure 5]d and [Figure 6]b.
Fine needle aspiration cytology was done for Case 4, and the diagnosis of chromoblastomycosis was missed initially. Smears showed hemorrhagic background with mixed inflammatory cells. Later the case was diagnosed in histological sections. On review, the cytology smears showed hemorrhagic background with scattered lymphocytes, plasma cells, clumps of erythrocytes, amidst there were scattered pale brown colored round structures (yeast cells) slightly larger than lymphocytes [Figure 3]. These structures showed equatorial septation [Figure 3]: inset.
| Discussion|| |
The term chromoblastomycosis is exclusively used for those slow growing chronic more or less granulomatous fungal infections which characteristically produced pigmented hard yeast cells called muriform cells or sclerotic bodies  and belong to the group of fungal infections called phaeohyphomycosis caused by dematiaceous (pigmented) fungi. Others in the group include superficial phaeohyphomycosis, subcutaneous phaeohyphomycosis and chromomycetoma.  Fonsecea pedrosoi is the commonest agent of chromoblastomycosis and the identification of specific etiological agents requires mycological culture studies. The fungus is highly prevalent in hot and humid climate and is ubiquitous found everywhere in soil, decaying vegetation and rotten wood which frequently served as the point source of infection. ,
The disease is slowly progressive, has varied clinical presentation ranging from ulcer to papulonodular to cauliflower like growth hence frequently confused clinically for other lesions.  The disease frequently involves lower extremities of barefooted adult rural men involved in agriculture related works. ,,, Other unusual sites of involvement include penile shaft, vulva, tonsils and ala of nose.  Non-healing ulcer in an 11-year-old boy indicates that the lesion is at its initial stage of evolution, and probably he would have been an adult by the time of presentation with classical tumorous mass. When fully evolved, the lesions are usually solitary or multiple with satellite lesions due to local spread and rarely the spread is hematogenous, when it occurs, the commonly isolated organism is Cladosporium Trichoides.
Diagnosis of chromoblastomycosis is easily made in Hand E stained sections when the organisms are in good numbers by demonstrating diagnostic 'Sclerotic bodies'. It is really challenging for pathologists when (1) the organisms are sparse, (2) the lesion is not fully evolved (as in early ulcer/ plaque stage) and (3) there is no 'clinical suspicion' in the request form. In the present study all six cases were clinically unsuspected and the diagnosis was incidental in histopathology. In this sense the only clue that made us search for organisms was giant cell granulomas, which were seen in majority of cases. In one case that presented as ulcer form has showed only microabscesses and the organisms were sparse. These difficult situations can be resolved by exploiting the property of 'natural pigmentation' of the etiologic agents provided there should be strong clinical suspicion of the lesion. This is achieved by screening both unstained sections and de stained sections (as well as smears) for diagnostic sclerotic bodies. Characteristic of chromoblastomycosis are natural pigmentation and reproduction by splitting (or septation) but not by budding. Because of their natural pigmentation, the sclerotic bodies can be easily identified in unstained sections and they retain their pigment even after 'de staining' the H and E stained sections (as well as smears). In the present study, sclerotic bodies were easily identified in both 'unstained' sections and 'de stained' sections without much difficulty in all six cases requiring relatively less time and the characteristic morphologic details such as septation and natural pigmentation of thick wall were well appreciated. These sclerotic bodies are brown round to polyhedral approximately 5- 8μm, seen against good contrast of colorless background in both unstained and de stained sections. They showed mature thick wall (just like outer border of copper penny) and characteristic intracellular septations. Though the splitting or septation in one or two planes is pathognomonic for the organism, not all organisms exhibit septations. Examining the unstained and de stained sections is easy, less cumbersome, time saving and cost effective than special stains and also removes the possibility of artifacts observed with special stains. Examination of de stained sections for sclerotic bodies after their detection in H and E stained sections provides the confirmation and avoids unnecessary special stains. De stained sections can be utilized for retrospective as well as prospective study of verrucous lesions for sclerotic bodies. However sclerotic bodies must be distinguished from frequently encountered brown colored artifacts (either hemosiderin or formalin derived) in tissue sections. Like sclerotic bodies, artifacts are also dense brown colored in both 'unstained' and H and E stained sections and retain their color even after de staining. The diagnostic sclerotic bodies have definite morphology with characteristic intracellular division (planate or triradiate) and 'copper penny' appearance. Artifacts are amorphous, irregular, dense brown colored and don't show characteristic division [Figure 5]f. Hence pseudoepitheliomatous hyperplasia, microabscesses and giant cell granulomas constitute important clues for pathologist to search for diagnostic sclerotic bodies in stained, unstained as well as in de stained sections.
The fungal elements of other subgroups in phaeohyphomycosis have varied morphology including hyphal forms, pseudohyphae, yeast forms, branching hyphal forms, spore forms, or in various combinations of above. They show variable melanin pigment on their walls and it depends on maturity and adaptation in the tissue. Early immature forms are less adapted and have little or no melanin pigment on them necessitating the need for special stains such as Gomori's methenamine silver stain and Fontana Masson to identify the fungus and the pigment respectively.  By definition, the diagnosis of chromoblastomycosis (a subgroup of phaeohyphomycosis) relies on the identification of 'sclerotic bodies' which always have brown colored thick wall (hence the name). And the fact that only those spore forms of phaeohyphomycosis which show characteristic brown colored thick wall are called as 'sclerotic bodies' or 'muriform cells' and are diagnostic of chromoblastomycosis. In this sense unstained preparations may be of little use in the diagnosis of other subgroups in Phaeohyphomycosis, while they are useful in the diagnosis of chromoblastomycosis. In contrast there is no role of special stains in the diagnosis of chromoblastomycosis, while they may be required in the diagnosis of other subgroups in phaeohyphomycosis. 
Commonly used special stain is Fontana Masson to demonstrate melanin pigment in the organism's wall [Figures 6]a-d. They are also useful for the illustration purpose, since they provide good contrast. , In the present study, Gomori's methenamine silver stain helped to delineate the hyphal forms in keratinized layers in Case 2 [Figure 4]c and d.
Since etiological agents of chromoblastomycosis look similar morphologically, histology has limitation in identifying the genus and the exact species, relying solely on cultural studies.
Tissue Response to Fungi [Table 2]
The most common tissue responses observed were pseudo-epitheliomatous hyperplasia with hyperkeratosis, keratinolytic micro-abscesses or pyogranulomas and giant cell granulomatous response. , In the present study, the consistent finding was micro abscess formation seen in all six cases where as epithelial hyperplasia and giant cell granulomatous response were observed in majority of cases. The appearance of typical verrucous lesion requires longer incubation period (possibly many months) during which etiologic agent undergoes adaptive change in the tissue responsible for characteristic hardness of sclerotic bodies. These sclerotic bodies remain dormant for many years and could explain the difficulty in achieving a cure in cases of chromoblastomycosis.  The hyphal forms are rare in chromoblastomycosis, if present it is often seen in dead horny layers of skin and may show branching and septation. The hyphal form is a feature of superficial chromohyphomycosis which usually presents as cutaneous pigmented macule with fungi inhabiting the dead cornified layers of skin.
Case 2 showed similar hyphal forms in cornified layers with scanty inflammatory cells. It is unclear whether hyphal forms are responsible for tissue changes seen in chromoblastomycosis or is it the infective form of fungus. It is observed that there are major changes in the cell wall content while adapting from hyphae to sclerotic bodies with rhamnose, palmitic, oleic and arachidonic acids being major composition in the latter as compared to glucose, mannose, galacto-fructose, glucosamine, lin-oleic acid and ergosterol in the former. 
Typically, verrucous lesions show small tiny black dots on the surface of each papillae. It is very important to note or look for such black spots on the verrucae because they represent microabscesses seen in histology and is ideal to sample from such site for histopathology or scrapings for cytological examination.  These microabscesses contain the pathogenic spores, which are in the process of elimination to the external environment by phenomenon called as transepithelial migration. Release of pathogenic sclerotic bodies by transepithelial migration and their autoinoculation and or lymphatic spread could explain the 'satellite' lesions seen in Case 3. It appears that transepithelial elimination is an important phenomenon responsible for pathophysiology and epidermal changes seen in chromoblastomycosis, , were observed in majority of cases. Transepithelial migration is also observed in other cutaneous lesions such as Rhinosporidiosis and perforating granuloma annulare. The epithelial responses are divided into three types based on the integrity of epithelium, size and quality of foreign particles eliminated. The first and second types show little or no histological disruption, whereas definite histological disruption is seen in the third type with acanthosis, hyper-parakeratosis and at times pseudo-epitheliomatous hyperplasia which are characteristic of fully evolved lesions of chromoblastomycosis. 
Cytological Diagnosis of Chromoblastomycosis
Review of literature showed only one case report of cytodiagnosis of chromoblastomycosis.  It reflects that many cases are missed cytologically (as well as histologically) due to lack of 'clinical suspicion'. Frequently the aspiration smears show mixed inflammatory cells and the fact that fungal spores look orange to reddish brown and are of the size of red cells. Red cells are pink and usually form rouleaux, whereas, sclerotic bodies are pale brown or brownish yellow color and usually occurs in singles in aspiration smears (occurrence in chains is a characteristic feature in tissue sections). Though septation is not a consistent feature, if present, it is a characteristic feature of chromoblastomycosis. In Case 4 there was a similar situation in that we missed the lesion cytologically, but could identify it histopathologically. On review, both H and E smears as well as 'de stained' smears showed characteristic sclerotic bodies.
In conclusion, unstained and de stained preparations are as efficient as special stains and more efficient than H and E preparation in the identification of sclerotic bodies. Whenever pathologists encounter warty, verrucous lesions they should make every effort for complete search for the diagnostic sclerotic bodies in both unstained and de stained sections. Since the clinical suspicion is very important in the diagnosis of chromoblastomycosis, clinician should include it in the differential diagnosis whenever they encounter above lesions and should send the unfixed extra sample for mycological culture studies.
| Acknowledgement|| |
We thank Dr JH Makannavar and Dr MH Kulkarni for the support they have given for the article.
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Sateesh S Chavan
Department of Pathology, Karnataka Institute of Medical Sciences, Hubli-580 022
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]