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ORIGINAL ARTICLE  
Year : 2014  |  Volume : 57  |  Issue : 2  |  Page : 217-222
Diagnostic utility of melanin production by fungi: Study on tissue sections and culture smears with Masson-Fontana stain


1 Department of Pathology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Andhra Pradesh, India
2 Department of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Andhra Pradesh, India

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Date of Web Publication19-Jun-2014
 

   Abstract 

Background: Dematiaceous fungi appear brown in tissue section due to melanin in their cell walls. When the brown color is not seen on routine H and E and culture is not available, differentiation of dematiaceous fungi from other fungi is difficult on morphology alone. Aims and Objective: To study if melanin production by dematiaceous fungi can help differentiate them from other types of fungi. Materials and Methods: Fifty tissue sections of various fungal infections and 13 smears from cultures of different species of fungi were stained with Masson Fontana stain to assess melanin production. The tissue sections included biopsies from 26 culture-proven fungi and 24 biopsies of filamentous fungi diagnosed on morphology alone with no culture confirmation. Results: All culture-proven dematiaceous fungi and Zygomycetes showed strong positivity in sections and culture smears. Aspergillus sp showed variable positivity and intensity. Cryptococcus neoformans showed strong positivity in tissue sections and culture smears. Tissue sections of septate filamentous fungi (9/15), Zygomycetes (4/5), and fungi with both hyphal and yeast morphology (4/4) showed positivity for melanin. The septate filamentous fungi negative for melanin were from biopsy samples of fungal sinusitis including both allergic and invasive fungal sinusitis and colonizing fungal balls. Conclusion: Melanin is produced by both dematiaceous and non-dematiaceous fungi. Masson-Fontana stain cannot reliably differentiate dematiaceous fungi from other filamentous fungi like Aspergillus sp; however, absence of melanin in the hyphae may be used to rule out dematiaceous fungi from other filamentous fungi. In the differential diagnosis of yeast fungi, Cryptococcus sp can be differentiated from Candida sp by Masson-Fontana stain in tissue sections.

Keywords: Dematiaceous, fungi, non-dematiaceous, Masson-Fontana, melanin

How to cite this article:
Sundaram C, Shantveer G U, Umabala P, Lakshmi V. Diagnostic utility of melanin production by fungi: Study on tissue sections and culture smears with Masson-Fontana stain. Indian J Pathol Microbiol 2014;57:217-22

How to cite this URL:
Sundaram C, Shantveer G U, Umabala P, Lakshmi V. Diagnostic utility of melanin production by fungi: Study on tissue sections and culture smears with Masson-Fontana stain. Indian J Pathol Microbiol [serial online] 2014 [cited 2018 Oct 22];57:217-22. Available from: http://www.ijpmonline.org/text.asp?2014/57/2/217/134666



   Introduction Top


Melanins are enigmatic pigments that defy exact characterization. They are produced by various micro organisms including pathogenic fungi. [1] Dematiaceous fungi contain melanin in their cell walls and appear brown in tissue sections. More than 100 species and 60 genera of dematiaceous fungi have been implicated in human disease, and some of the common agents include Alternaria, Bipolaris, Cladophialophora, Cladosporium, Curvularia, Exophiala, Fonsacea species. [2],[3] They cause cerebral, subcutaneous, and other deep-seated infections in young and often immunocompetent hosts. These infections require aggressive treatment and are associated with poor outcome. [4],[5] Diagnosis of these infections requires histopathologic examination and culture. Dematiaceous fungi show pigmented hyphae on histology; however, the degree of pigmentation may vary. [6] The diagnosis becomes difficult when the brown color of these fungi is not apparent on routine Hematoxylin and eosin (H and E)-stained sections and when culture is not available. They also have to be differentiated from other filamentous fungi including Aspergillus sp, hyalohyphomycetes (Scedosporium, Fusarium, Paecilomyces species) and the mucorales group of fungi (Mucor, Rhizopus, Rhizomucor, Absidia, Cunninghamella species), which is important for therapy and prognosis. [7],[8] Fungal morphology, tissue reaction on histology, and isolation of the organism in culture complement one another in the identification of fungi. Culture studies may not be available all the time either because diagnosis is not suspected clinically/material not submitted for culture or due to lack of facilities. Sometimes, the organisms fail to grow in culture even when the material is submitted. In such circumstances, the diagnosis is based on tissue sections alone. However, diagnosis on morphology alone has certain limitations. A lack or scarcity of fungal pigmentation, fixing the specimen without prior culture, use of routine microscopic procedures, and H and E stain make the diagnosis of dematiaceous difficult. [9] Hence, we plan to study if melanin production by dematiaceous fungi can help in the differentiating them from other types of fungi.


   Materials and methods Top


Fifty formalin-fixed paraffin-embedded tissue sections diagnosed as fungal infections from brain, paranasal sinus (PNS), bone, lung, stomach, skin, and soft tissues were included in the study. All the paraffin blocks were less than 10 years old. The tissue sections included 26 biopsies of culture-proven fungi and 24 biopsies with no culture confirmation. The fungi showing thin septate hyphae with acute angle branching and no brown color on H and E and no culture confirmation were classified as septate filamentous fungi. The fungi with broad aseptate hyphae and branching irregularly or at right angles and no culture confirmation were classified as Zygomycetes. The fungi with both hyphal and yeast forms, irregular branching and no brown color on H and E with no culture confirmation were classified as filamentous septate fungi with non-specific features. All the sections were stained with H and E to assess tissue reaction and pathology, Gomori's methenamine silver (GMS) and periodic acid Schiff (PAS) to delineate fungal morphology and Masson Fontana (MF) stain to assess melanin production. Melanin positivity was graded as strong, weak, or negative based on intensity. Mucicarmine and alcian PAS stains were done wherever necessary. Thirteen smears from cultures of various species of Aspergillus, Zygomycetes, Cryptococcus, Candida, and dematiaceous fungi were fixed in absolute alcohol and stained with MF to assess melanin production in vitro.


   Results Top


[Table 1] gives the type of fungus with source of tissue, pathology, and result of staining with MF on tissue sections. [Table 2] shows the type of fungus and the result of staining with MF on smears from culture. In tissue sections, all samples of Aspergillus flavus showed positivity with MF [Figure 1]a-c] with variable intensity, whereas Aspergillus fumigatus showed strong positivity in one [Figure 1]d] and was negative in two. Smears from cultures of Aspergillus niger, terreus, nidulans, and fumigatus showed positivity of hyphae, fruiting heads, and conidia with MF [Figure 2]a-d], whereas Aspergillus flavus showed positivity of fruiting heads and conidia only with hyphae being negative with MF [Figure 2]e].
Figure 1: Aspergillus in tissue section. (a) Giant cells with intracytoplasmic negatively staining hyphae (H and E; ×400). (b) Narrow septate branching hyphae within and outside the giant cells (GMS; ×400). (c) Positive staining of Aspergillus flavus hyphae with MF stain (MF stain; ×400). (d) Positive staining of Aspergillus fumigatus hyphae with MF stain (MF stain; ×400)

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Figure 2: Masson-Fontana staining on culture smears of Aspergillus. (a) Aspergillus fumigatus, (b) Aspergillus terreus, (c) Aspergillus nidulans, (d) Aspergillus niger showing strong positivity of hyphae and conidia (MF stain; ×200). (e) Aspergillus flavus showing positivity of conidia with hyphae being negative (MF stain; ×200)

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Table 1: Masson-Fontana staining on tissue sections

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All genera of culture-proven Zygomycetes in tissue sections including Rhizopus oryzae [Figure 3]a-c](3), Apophysomyces elegans (2), and Basidiobolus ranarum (2) showed strong positivity with MF. In culture smears, Rhizopus oryzae showed strong positivity [Figure 3]d] and Absidia corymbifera was weakly positive [Figure 3]e].
Figure 3: Zygomycetes in tissue section and culture smears. (a) Angioinvasion by faintly basophilic broad, aseptate irregularly branching hyphae of Rhizopus (H and E; ×200). (b) Broad, aseptate irregularly branching hyphae of Rhizopus sp highlighted by silver stain (GMS stain; ×400). (c) Positive staining of Rhizopus sp with MF stain in tissue section (GMS stain; ×400). (d) Rhizopus sp showing strong positivity and e, Absidia sp showing positivity with MF stain on culture smears (MF stain; ×400)

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All samples of Cryptococcus neoformans in tissue sections [Figure 4]a-d] (3) and culture smear showed strong positivity for MF, whereas both species of Candida i.e. albicans and tropicalis showed negative staining in tissue sections [Figure 5]a-d] and culture smears [Figure 5]e and f].
Figure 4: Cryptococcus in tissue section. (a) Refractile budding yeast forms (H and E; ×400). (b) Mucicarmine stain and (c) Alcian-PAS stain highlighting the mucoid capsule (×200). (d) Strong positive staining with MF stain (MF stain; ×400)

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Figure 5: Candida in tissue sections and culture smears. (a) Basophilic staining yeast and pseudo hyphae in tissue section (H and E; ×200). (b) PAS stain and (c) GMS stain highlighting the fungi in tissue section (×200). (d) Negative staining with MF stain in tissue section (×200). (e) Candida albicans and (f) Candida tropicalis showing negative staining with MF stain in culture smears (MF stain; ×400)

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All the culture-proven hyalohyphomycetes including Pseudallescheria boydii (1) [Figure 6]a and b], and dematiaceous fungi including Nodulosporium (1), Cladophialophora bantianum (1), and Sporothrix scheinkii (1) showed strong positivity with MF in tissue sections. The same was true for culture smears of Curvularia lunata [Figure 6]c], Cladophialophora bantianum [Figure 6]d], and Fonsacea pedrosoi [Figure 6]e]. Except for Cladophialophora bantianum, others did not show brown color on H and E sections.
Figure 6: (a) Faintly basophilic narrow septate branching hyphae of Pseudallescheria boydii in tissue section (H and E; ×400). (b) Positive staining of Pseudallescheria boydii with MF stain in tissue section (H and E; ×200). (d) Curvularia lunata, (e) Cladophialophora bantiana and (f) Fonsacea pedrosoi showing strong positivity with MF stain on culture smears (MF stain; ×400)

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Table 2: Masson-Fontana staining on culture smears

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Nine of the 15 filamentous septate fungi, 4/5 Zygomycetes, and 4/4 filamentous fungi hyphal and yeast forms with features on morphology in tissue sections showed positivity for melanin. The six filamentous septate fungi and one sample of Zygomycetes which were negative for melanin were from PNS biopsies including both allergic and invasive sinusitis and colonizing fungal balls.


   Discussion Top


Dematiaceous fungi cause a variety of cutaneous, subcutaneous, and invasive or disseminated infections in immunocompetent and immunocompromised hosts. [8],[10] They are neurotropic and cause brain abscess and sinusitis with extension to brain. [10] The diagnosis of dematiaceous fungal infections can be difficult. Identification in culture requires expertise, and currently there are no reliable molecular methods for diagnosis. [11] In tissues, they have a characteristic appearance of thin, irregularly swollen hyphae with prominent septae that show constrictions and yeast-like structures in contrast to Aspergillus species, which typically show septate, acutely branching, straight-walled hyphae. [11] The hyphae have melanin in their cell walls, which imparts a dark brown color to them. Masson Fontana stain was used to accentuate weak pigmentation of melanin in the cell walls of dematiaceous fungi. [9],[12] Subsequently, when culture studies were not available, MF stain was believed to be useful to differentiate dematiaceous fungi from other filamentous fungi, especially in allergic fungal sinusitis. [13],[14]

Diagnosis of fungal infection starts with H and E; GMS and PAS stains are used for screening for fungal hyphae and yeast forms. GMS stain is very useful to delineate fungal hyphae but can cause diagnostic problems as it stains normal structures like collagen, basement membrane, and other silver-positive filamentous structures. GMS also stains neurosecretory granules and melanin, which cause problems in differentiating them from yeast forms. PAS stains fungal walls as they contain glycogen. Combination of H and E with GMS or PAS helps in colocalizing the normal structures and fungal hyphae or yeast forms. [6] Masson-Fontana stains melanin in fungal walls but also stains melanin in the skin. [6] Kimura and McGinnis in their study of 53 culture-proven fungal infections on tissue sections stained with MF concluded that MF can stain fungi other than dematiaceous fungi. [15] [Table 3]. gives the comparison of results of MF staining between the present study and the study by Kimura and McGinnis. [15] The present study tested the production of melanin in culture also by staining smears from culture with MF as melanin production may vary between in vitro and in vivo conditions. The results of the present study on tissue sections are essentially similar to the observations made by Kimura and McGinnis with minor variations. [15] Aspergillus sp showed variable results in the staining of hyphae, fruiting heads, and conidia, (especially fumigatus and flavus species), between tissue sections and culture smears. Studies on melanin production by Aspergillus fumigatus showed that melanin was confined to conidial structures and was absent from hyphae. [16] Fruiting heads and condia of Aspergillus sp are not seen in tissue sections (except rarely in cavitary lesions of lungs / PNS), and this explains the discrepancy in the staining patterns between culture smears and tissue sections.
Table 3: Comparison of results of the present study with those of Kimura and McGinnis

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Studies on the temporal expression of melanin production during in vitro culture of Aspergillus fumigatus showed that melanization was induced during infection. This may explain the positivity of hyphae and fruiting heads of Aspergillus fumigatus and other species in culture and the variable expression in tissue sections in the present study. Lack of melanization in Aspergillus fumigatus was shown to be due to defective genes involved in melanin production. [17] Fungi lacking pigment were shown to be more susceptible to attack by monocytes, polymorphonuclear leukocytes, and reactive oxygen species. [18]

The genera of Zygomycetes tested in the present study included Absidia, Rhizopus, and Basidiobolus. All of them showed positivity in tissue sections and culture, though Absidia corymbifera showed weak staining intensity. These results were in agreement with the study by Kimura and McGinnis. [15] A study comparing melanizing and non-melanizing strains of Basidiobolus sp found that the melanized isolates were associated with human disease. [19]

In the present study, Cryptococcus sp were positive and Candida sp were negative for melanin in both culture smears and tissue sections. Hence, MF stain can be used in the differential diagnosis of Cryptococcus sp and Candida sp in tissue sections. All the dematiaceous fungi showed positivity for melanin on MF in tissue sections and culture smear. Only one biopsy of Cladophialophora bantianum showed brown color on tissue sections. The pigmented hyphae are diagnostic of dematiaceous fungi in tissue sections on H and E as Aspergillus sp and other hyaline fungi do not show melanin on routine H and E in tissue sections. All the fungi with dimorphic morphology in tissue section and no culture confirmation were positive for melanin. The irregularly swollen septate hyphae with round or oval yeast forms were better visualized with MF stain, and the diagnosis may be confirmed as dematiaceous fungi. Similar observations were made earlier. [9],[14]

There was variable positivity for melanin for the septate filamentous fungi, which had no culture confirmation. Six of the septate fungi and one aseptate fungus were negative for melanin. All these biopsies were from fungal sinusitis samples, which included both allergic and invasive sinusitis and colonizing fungal balls. The etiological agents of fungal sinusitis include Aspergillus sp. Zygomycetes, and other hyaline septate fungi and dematiaceous fungi. [20],[21],[22] The results of the present study emphasize that MF stain alone cannot differentiate dematiaceous from non-dematiaceous fungi. However, absence of melanin in the hyphae of filamentous fungi can be used in differentiating dematiaceous from other filamentous fungi.

Melanin production is linked to virulence by promoting survival in the host, thus allowing the organism to cause disease and also to produce intense inflammatory response that results in host damage. [1] Studies on melanization of Cryptococcus neoformans show that melanin protects the organism against antifungal agents like Amphoterecin B and Caspofungin. [23],[24] Melanin is required for the correct assembly of different layers for the conidial wall in Aspergillus fumigatus, thus facilitating expression of adhesions and other virulence factors at the candidial surface. [25] Thus, melanin production may not help differentiate various types of fungi but may contribute to the pathogenicity of fungi and their antifungal susceptibility.


   Conclusion Top


Melanin is produced by both dematiaceous and non-dematiaceous fungi. Masson-Fontana stain cannot be used to reliably differentiate dematiaceous fungi from Aspergillus sp and other filamentous fungi; however, in the differential diagnosis of septate fungi, absence of melanin in the cell walls assessed by MF stain can be used to rule out dematiaceous fungi. The MF stain can be used to differentiate Cryptococcus sp from Candida sp in the tissue sections. There is variability of production of melanin in in vivo and in vitro conditions, which may be related to infection and culture conditions.

 
   References Top

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4.Brandt ME, Warnock DW. Epidemiology, clinical manifestations, and therapy of infections caused by dematiaceous fungi. J Chemother 2003;15Suppl 2:36-47.   Back to cited text no. 4
    
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23.Wang Y, Casadevall A. Growth of Cryptococcus neoformans in presence of L-dopa decreases its susceptibility to amphotericin B. Antimicorb Agents Chemother 1994;38:2648-50.  Back to cited text no. 23
    
24.vanDuin D, Casadevall A, Nosanchuk JD. Melanization of Cryptococcus neoformans and Histoplasmacapsulatum reduces their susceptibility to amphoterecin B and caspofungin. Antimicrob Agents Chemother 2002;46:3394-400.  Back to cited text no. 24
    
25.Pihet M, Vandeputte P, Tronchin G, Renier G, Saulnier P, Georgeault S, et al. Melanin in an essential component for the integrity of the cell wall of Aspergillusfumigatus conidia. BMC Microbiol 2009;9:177.  Back to cited text no. 25
    

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Correspondence Address:
Challa Sundaram
Senior Professor, Department of Pathology, Nizam's Institute of Medical Sciences, Hyderabad - 500 082, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.134666

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