Novel Application of the Masson-Fontana Stain for Demonstrating Malassezia Species Melanin-Like Pigment Production In Vitro and in Clinical Specimens

Abstract

Melanin-like pigment produced in vitro and in vivo byMalassezia yeasts has not been described before. Masson-Fontana staining confirmed accumulation of black pigment on the cell walls of L-dihydroxyphenylalaline (L-DOPA)-cultured Malassezia species. Black pigment was also observed in cells and hyphae from hyperpigmented patient lesions with culture-confirmed pityriasis versicolor and seborrheic dermatitis.Melanin production, mediated by a copper-containing phenoloxidase enzyme using L-dihydroxyphenylalaline (L-DOPA) as a substrate, is a well-studied pathogenetic mechanism in fungi (9, 24), especially in species of the human pathogen Cryptococcus. Many properties related to the evasion of the host immune system (9) and antifungal drug resistance (23) have been attributed to melanin or melanin-like pigments, assessed in vitro by growth in L-DOPA agar (18) and in vivo by staining with Masson-Fontana stain, which detects melanin deposited on the cells of this basidiomycetous yeast (10).The aims of this study were (i) to assess the in vitro abilities of Malassezia yeasts to oxidize L-DOPA and produce a melanin-like pigment and (ii) to demonstrate that this pigment can be detected in yeast cells and hyphae by Masson-Fontana silver staining of skin scales from pityriasis versicolor (PV) and seborrheic dermatitis (SD) patients.We studied 53 type, reference, and clinical isolates of 11 Malassezia species (Table 1) (7,8,(20)(21)(22). The type and reference strains for this study were obtained from the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. The clinical isolates were strains kept in the Mycology Reference Laboratory of the Hellenic National Collection of Pathogenic Fungi (HNCPF; World Data Centre on Microorganisms member type ID 2023). All Malassezia strains, the control Cryptococcus grubii strains (HNCPF 6417a and 6417b) demonstrating maximum and no melanization, respectively, and the autoch- The solidified agar surface was punctured with a standard sterile 5-mmdiameter cork borer, producing wells. In each well, 100 l of whole-cell suspensions or cells that had been aseptically mechanically disrupted by an orbital homogenizer at 100 rpm and suspended in sterile water was inoculated and incubated at 35°C for 3 and 7 days for the Cryptococcus and Malassezia strains, respectively (Fig. 1A and 2B). All plates were inspected daily for pigment production. Absence of a lipid source in the culture medium did not allow growth of Malassezia yeasts.The Masson-Fontana silver stain was employed to demonstrate melanin deposition (10) on the walls of Malassezia cells harvested from tyrosine and L-DOPA media, respectively. In order to ascertain the necessity for L-DOPA in the synthesis of melanin, cells grown in the L-DOPA-depleted medium were also tested for melanin-like pigment detection by Masson-* Corresponding author. Mailing address: Mycology Reference Laboratory,

Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus

“…Therefore, based on the present analysis, none of the Malassezia species should have levels of melanin production comparable to those of C. neoformans , with the possible exception of M. cuniculi . Corroborating this statement, Gaitanis and colleagues (37) demonstrated for several Malassezia species the ability to produce melanin on l -DOPA but at lower rate than those of C. neoformans and C. gattii .…”

“…These findings are in contrast with those of Youngchim and colleagues (25), who observed melanin synthesis by strain NBRC 0656 of M. furfur on l -DOPA, medium conditions which also induced the production of hypha-like forms not observed for strains CBS 14141 and CBS 7982. However, in another study, Gaitanis and colleagues showed that more than 10 strains of M. furfur displayed little or no melanin production on l -DOPA medium (37), indicating that strains that belong to the same species, M. furfur , can be characterized by relevant phenotypic differences. As regards the melanin production of M. furfur NBRC 0656, it seems that this strain is related to the M. furfur hybrid CBS 1878 (Teun Boekhout, personal communication), and hence its ability to produce melanin could be due to the combined activity of two LAC2 genes.…”

The Malassezia Genus in Skin and Systemic Diseases

“…However, conventional culture and identification methods offer the advantage of further evaluating the isolates for possible virulence factors, such as the production of phospholipase (44,170) and indole (108,184,336) and melanin synthesis (107). Furthermore, this was highlighted in a study by Akaza et al (6), in which the seasonal rates of isolation of Malassezia species from healthy skin determined by quantitative PCR (qPCR) were compared with those determined by use of Leeming-Notman agar (197).…”

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment

“…The nuclear area was calculated by MATLAB software (The MathWorks Inc., Natick, Massachusetts, USA) and summed as the mean±SD for statistical analysis. Fontana–Masson stain and iron stain (hemosiderin) (International Medical Equipment Inc., Oceanside, California, USA) followed routine protocols [5,6]. …”