George Gaitanis

Use of Fatty Acid RPMI 1640 Media for Testing Susceptibilities of Eight Malassezia Species to the New Triazole Posaconazole and to Six Established Antifungal Agents by a Modified NCCLS M27-A2 Microdilution Method and Etest

A novel formulation of RPMI 1640 medium for susceptibility testing of Malassezia yeasts by broth microdilution (BMD) and Etest is proposed. A modification of the NCCLS M27-A2 BMD method was used to test 53 isolates of Malassezia furfur (12 isolates), M. sympodialis (8 isolates), M. slooffiae (4 isolates), M. globosa (22 isolates), M. obtusa (2 isolates), M. restricta (2 isolates), M. pachydermatis (1 isolates), and M. dermatis (2 isolates) against amphotericin B, ketoconazole, itraconazole, fluconazole, voriconazole, terbinafine, and posaconazole by BMD and Etest. RPMI and antibiotic medium 3 (AM3) were supplemented with glucose, bile salts, a mixture of fatty acids, and n-octadecanoate fatty acids and Tween 20. M. furfur ATCC 14521 and M. globosa ATCC 96807 were used as quality control strains. Depending on the species, MICs were read after 48 or 72 h of incubation at 32 degrees C. Low azole and terbinafine MICs were recorded for all Malassezia species, whereas amphotericin B displayed higher MICs (>/=16 microg/ml) against M. furfur, M. restricta, M. globosa, and M. slooffiae strains, which were AM3 confirmed. Agreement of the two methods was 84 to 97%, and intraclass correlation coefficients were statistically significant (P < 0.001). Because of higher amphotericin B MICs provided by Etest for strains also displaying high BMD MICs (>/=1 microg/ml), agreement was poorer. The proposed media are used for the first time and can support optimum growth of eight Malassezia species for recording concordant BMD and Etest MICs.

The range of molecular methods for typing Malassezia.

Purpose of review The recent sequencing of the whole genome of Malassezia globosa and M. restricta forms the basis for molecular epidemiology studies and instigates investigations into their respective virulence factors. Thus, reviewing current knowledge on Malassezia molecular typing methods would reveal the pros and cons of each method and would highlight potential scarcity of epidemiological data regarding this ubiquitous fungal commensal and pathogen. Recent findings Methods employed for Malassezia molecular typing can be categorized into those detecting sequence variations of strains and those that selectively amplify polymorphic DNA markers for discriminating Malassezia species subtypes. The former exploit rRNA gene sequence variations in order to trace M. globosa, M. restricta and M. pachydermatis subtypes associated with specific skin diseases, or detect M. furfur geographical variations. Polymorphic DNA amplification methods, such as amplified fragment length polymorphism analysis, demonstrated association of M. furfur subtypes with the origin of the strain (skin or systemic isolate), whereas PCR-fingerprinting of the mini-satellite DNA clustered M. furfur strains according to their geographic origin and disease origin. Moreover, much typing work has already been performed regarding the zoophilic species M. pachydermatis and the relevant methods can be adapted for studying the anthropophilic Malassezia species. Summary In the near future, molecular typing will be a powerful tool in epidemiological studies that could be employed for the elucidation of the pathobiology of Malassezia species in associated skin diseases.

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 by Malassezia 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.

Clinical expression and new SPINK5 splicing defects in Netherton syndrome: unmasking a frequent founder synonymous mutation and unconventional intronic mutations.

Netherton syndrome (NS) is a severe skin disease caused by loss-of-function mutations in SPINK5 (serine protease inhibitor Kazal-type 5) encoding the serine protease inhibitor LEKTI (lympho-epithelial Kazal type-related inhibitor). Here, we disclose new SPINK5 defects in 12 patients, who presented a clinical triad suggestive of NS with variations in inter- and intra-familial disease expression. We identified a new and frequent synonymous mutation c.891C>T (p.Cys297Cys) in exon 11 of the 12 NS patients. This mutation disrupts an exonic splicing enhancer sequence and causes out-of-frame skipping of exon 11. Haplotype analysis indicates that this mutation is a founder mutation in Greece. Two other new deep intronic mutations, c.283-12T>A in intron 4 and c.1820+53G>A in intron 19, induced partial intronic sequence retention. A new nonsense c.2557C>T (p.Arg853X) mutation was also identified. All mutations led to a premature termination codon resulting in no detectable LEKTI on skin sections. Two patients with deep intronic mutations showed residual LEKTI fragments in cultured keratinocytes. These fragments retained some functional activity, and could therefore, together with other determinants, contribute to modulate the disease phenotype. This new founder mutation, the most frequent mutation described in European populations so far, and these unusual intronic mutations, widen the clinical and molecular spectrum of NS and offer new diagnostic perspectives for NS patients.

Malassezia furfur fingerprints as possible markers for human phylogeography

Malassezia furfur was the first species described within the cosmopolitan yeast genus Malassezia, which now comprises 13 species. Reported isolation rates of these species from healthy and diseased human skin show geographic variations. PCR-fingerprinting with the wild-type phage M13 primer (5′-GAGGGTGGCGGTTCT-3′) was applied to investigate phylogeographic associations of M. furfur strains isolated from Scandinavians residing permanently in Greece, in comparison to clinical isolates from Greek, Bulgarian and Chinese native residents. Seven M. furfur strains from Scandinavians were compared with the Neotype strain (CBS1878), CBS global collection strains (n=10) and clinical isolates from Greece (n=4), Bulgaria (n=15) and China (n=6). Scandinavian, Greek and Bulgarian M. furfur strains mostly formed distinct group clusters, providing initial evidence for an association with the hosts geographical origin and with the underlying skin condition. These initial data address the hypothesis that M. furfur could be a eukaryotic candidate eligible for phylogeographic studies.

Malassezia Yeasts in Seborrheic and Atopic Eczemas

Malassezia yeasts are implicated in the pathogenesis of two common diseases: seborrheic and atopic eczema. Seborrheic eczema (SE) affects areas of the body with an increased density of sebaceous glands (scalp, mid eyebrow, nasolabial folds, sternum and interscapular regions) and body folds). M. globosa and M. restricta are more commonly isolated in lesional skin, but this is also true for the isolation rate from healthy skin. In lesions of SE, a nonimmunogenic irritant reaction is recognized by immunocytochemistry. SE-specific molecular subtypes of M. globosa, M. restricta, and M. furfur have been recognized. Production of potent aryl hydrocarbon receptor ligands by Malassezia yeasts could offer new insight in the pathogenesis of SE through modulation of the immune system. The persistent and recurring nature of SE is highlighted through the existence of multiple, but not yet curative topical and systemic therapies. Atopic eczema (AE) has variable clinical presentation of skin lesions depending on the age of the patient. Three types of AE are recognized: “extrinsic,” “intrinsic,” and one-third group of patients with extrinsic or intrinsic AE showing IgE-mediated sensitization to self-antigens. Malassezia yeasts have been shown to act as allergens in AE, and thirteen allergens have been cloned, characterized, and produced as recombinant proteins from Malassezia species. Some of these recombinant allergens show a high degree of sequence identity to homologous human self antigens. The human proteins are capable of inducing positive skin prick and atopy patch tests in patients sensitized to the Malassezia proteins, indicating a role of IgE-mediated autoreactivity in the pathogenesis of AE in a subset of patients. The recombinant allergens will contribute to improve the diagnosis of sensitization to the yeast in AE patients. A correct patient stratification according to specific sensitization patterns could open novel possibilities for an immunotherapeutic treatment of a subset of AE patients in the future.

Physiology and Biochemistry

Due to the ecologic niche of Malassezia yeasts, host-specific adaptations are an important issue in their biology. The commensal status of Malassezia yeasts is not clearly distinguished from the pathogenic stage, as transition from the one to the other is probably a continuum and not an on/off condition. Part of this pathogenic potential is determined by the activities of the enzymatic systems of Malassezia yeasts. The efficiency with which Malassezia yeasts utilize nutrients on the skin surface and in the sebaceous gland determines the size of their population, and also the quality and quantity of the produced metabolic by-products. This organic material ranges from free fatty acids that apply their action through an irritating or toxic effect to highly bioactive indole derivatives that bind to specific cellular receptors and regulate the expression of downstream metabolic pathways. The recently reported genome and secretory proteome of M. globosa and, in part, of M. restricta provide a molecular basis to understand the adaptions of Malassezia yeasts to their environment and to identify factors of pathogenicity. As many experiments on Malassezia biochemistry and physiology had been performed before the current taxonomy of Malassezia species was developed, and the corresponding strains were not deposited in official fungal collections, the genus name Malassezia will be used to include strains that were categorized as Malassezia furfur (sensu lato), Pityrosporum ovale and Pityrosporum orbiculare (see Chap. 2).

Immunocryosurgery for patients with therapeutically challenging basal cell carcinomas: Report of two representative cases

Surgery, including Mohs’ technique, is regarded the reference modality for the treatment of most basal cell carcinomas (BCCs) (1). Yet surgical treatment in challenging cases (large, multiple, confluent or recurrent BCCs) can be an arduous undertaking with relatively high relapse rates, particularly when conventional surgery is employed. ‘Immunocryosurgery’ has been recently introduced as a non-surgical, tissuesparing combination of cryosurgery during ongoing topical imiquimod for the treatment of ‘nonsuperficial’ BCCs (2). Herein, on the occasion of two cases, we would like to highlight the efficacy of immunocryosurgery as a treatment alternative for patients with challenging BCCs: (i) multiple/concurrent facial tumors that require extensive surgery and (ii) BCC relapses that would have mandated major reconstructive headand-neck surgery. Patient 1: A 71-year-old farmer presented with multiple (n = 18) facial BCCs (Figure 1A, B). Pathology confirmed diagnosis in two excised (eyelid BCCs) and six biopsied tumors. His medical history included prostate cancer, coronary heart disease and hyperlipidemia on goserelin acetate, clopidogrel and simvastatin, respectively. Excessive sun exposure was his only risk factor for multiple BCC. Nevoid BCC syndrome was excluded based on history, physical examination and assessment of relevant radiological signs. Daily 5% imiquimod cream (Aldara; Meda) was applied every evening on the nasal and left parietal lesions. After 2 weeks, no inflammation was evident and 0.1% tazarotene gel (Zorac; Pierre Fabre) was added every morning. Two weeks later (Figure 1C), a mild, open spray liquid N2 cryosurgery was applied to the inflamed BCCs (two freeze– thaw cycles (FTCs); 15-second freezing time each) and tazarotene and imiquimod were continued for an additional 4 weeks after cryosurgery. In this way, 16 tumors were treated in groups of three to five tumors and within 4 months the patient was diseasefree. During the 24-month follow-up, one tumor recurred (Figure 1D, arrow). Conclusively, in this patient, 15/16 tumors were effectively treated with one cycle of immunocryosurgery. Patient 2: A 63-year-old male was referred for a neglected, 28 17-mm, biopsy-proven recurrent BCC on his right ear 5 years after surgical removal of the primary (Figure 1E). His medical history included type II diabetes on glimepiride, arterial hypertension and coronary heart disease on olmesartan and metoprolol. After 3 weeks on daily imiquimod, liquid N2 cryosurgery was performed (open spray, two FTCs, 15-second freezing time each) followed by 3 additional weeks of imiquimod. The tumor cleared and has remained relapse-free for 18 months (Figure 1F).