Part 1: Understanding the role of Malassezia spp. in skin disorders: Malassezia yeasts as commensal or pathogenic organisms of human and animal skin.

INTRODUCTION: Malassezia spp. are a group of lipid-dependent basidiomycetes yeasts acting as commensal organisms of the human and animal skin. However, under some not well-defined circumstances, these yeasts may switch to opportunistic pathogens triggering a number of skin disorders with different clinical presentations. The genus comprises of 18 lipid-dependent species with a variable distribution in the hosts and pathologies thus suggesting a host- and microbe-specific interactions. AREA COVERED: This review highlighted and discussed the most recent literature regarding the genus Malassezia as a commensal or pathogenic organisms highlighting Malassezia-associated skin disorders in humans and animals and their antifungal susceptibility profile. A literature search of Malassezia associated skin disorders was performed via PubMed and Google scholar (up to May 2023), using the different keywords mainly associated with Malassezia skin disorders and Malassezia antifungal resistance. EXPERT OPINION: Malassezia yeasts are part of the skin mycobiota and their life cycle is strictly associated with the environment in which they live. The biochemical, physiological, or immunological condition of the host skin selects Malassezia spp. or genotypes able to survive in a specific environment by changing their metabolisms, thus producing virulence factors or metabolites which can cause skin disorders with different clinical presentations.

Differential lipase virulence in Malassezia furfur dimorphism isolated from pityriasis versicolor patients and healthy individuals.

BACKGROUND: Malassezia furfur is a member of the human skin microbiomes that can cause various skin diseases. Dimorphism plays a role as the yeast phase predominates during skin colonisation whereas mycelial forms are observed in the scales of patients with pityriasis versicolor (PV). However, due to their condition-dependence for growth, it is difficult to culture M. furfur and this is an additional challenge for studying the pathogenicity of this fungus. OBJECTIVE: To describe different media suitable for culturing Malassezia from the yeast phase into mycelial forms, with a particular focus on nutritional supplements and pH conditions. METHODS: Clinical M. furfur isolates from patients with PV and healthy individuals were used to investigate Malassezia dimorphism as well as the activity and expression of lipase enzymes. RESULTS: Our experimental media were significantly more likely to promote mycelial growth in strains from healthy individuals compared to those from patients with PV. Lipase activity was increased in the mycelial phase cells compared to yeast forms for all strains tested. Assessment of the relative transcriptional expression of lipase within M. furfur revealed that LIP-coding genes were upregulated in mycelium relative to yeast forms for the strains tested. However, the increases in LIP3, LIP5 and LIP6 gene expressions were significantly greater in strains from healthy individuals compared to those from patients with PV. CONCLUSION: Overall, this study validated effective growth conditions to study M. furfur virulence factors and demonstrated that lipase is associated with M. furfur dimorphism.

[Malassezia Display a Hyphae-like "Spaghetti-and-Meatballs" Configuration in Keratotic Plugs].

Malassezia are lipophilic yeasts in the skin microbiome that abundantly colonize all parts of human skin except for the soles of the feet. Fungal microbiome analysis of keratotic plugs from the noses of 10 healthy individuals identified Malassezia restricta as the predominant species, followed by Malassezia globosa. Malassezia hyphae were observed in 5 of the 10 individuals. The hyphae were curved and thick-walled with spherical thick-walled and grouped blastoconidia, described as a "spaghetti-and-meatballs" configuration. In this study, we observed Malassezia hyphae in keratotic plugs of healthy subjects, although abundant Malassezia hyphae have previously only been observed in lesional sites of patients with pityriasis versicolor.

Virulence factors of Malassezia strains isolated from pityriasis versicolor patients and healthy individuals.

Over the last decade, Malassezia species have emerged as increasingly important pathogens associated with a wide range of dermatological disorders and bloodstream infections. The pathogenesis of Malassezia yeasts is not completely clear, but it seems to be strictly related to Malassezia strains and hosts and needs to be better investigated. This study aimed to assess the enzymatic activities, biofilm formation and in vitro antifungal profiles of Malassezia spp. from pityriasis versicolor (PV) and healthy patients. The potential relationship between virulence attributes, the antifungal profiles and the origin of strains was also assessed. A total of 44 Malassezia strains isolated from patients with (n = 31) and without (n = 13) PV were employed to evaluate phospholipase (Pz), lipase (Lz), and hemolytic (Hz) activities and biofilm formation. In addition, in vitro antifungal susceptibility testing was conducted using the CLSI broth microdilution with some modifications. A high percentage of strains produced Pz, Lz, Hz and biofilm regardless of their clinical origin. The highest number of strains producing high enzymatic activities came from PV patients. A correlation between the intensity of hydrolytic activities (Lz and Pz activities) and the Hz activity was detected. Positive associations between Lz and the low fluconazole susceptibility and Hz and biofilm formation were observed. These results suggest that enzyme patterns and biofilm formation along with antifungal profiles inter-play a role in the pathogenicity of Malassezia spp. and might explain the implication of some Malassezia spp. in invasive fungal infections and in the development of inflammation. LAY SUMMARY: There is still little information on the virulence factors of Malassezia spp., despite their implication in severe diseases. Phospholipase, lipase, and hemolytic activities, biofilm formation and decreased antifungal susceptibility seem to contribute to their virulence in susceptible hosts.

Malassezia virulence factors and their role in dermatological disorders.

Malassezia is a commensal fungus that constitutes normal skin microbiota. However, in certain conditions and individuals, it may transform into a pathogenic yeast with multiple associated dermatological disorders and various clinical manifestations. This phenomenon is influenced by a unique host-agent interaction that triggers the production of several virulence factors, such as indoles, reactive oxygen species, azelaic acid, hyphae formation, and biofilm formation. This review article discusses Malassezia virulence factors that contribute to the transformation of Malassezia from commensal to pathogenic as well as their role in dermatological disorders, including pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, atopic dermatitis, and psoriasis.

Effectiveness of FastFung agar in the isolation of Malassezia furfur from skin samples.

BACKGROUND: Lipophilic basidiomycetous yeasts of the Malassezia genus can cause various skin diseases, such as seborrheic dermatitis, pityriasis versicolor, folliculitis and atopic dermatitis, and even life-threatening fungemia in newborns and immunocompromised individuals. Routine mycological media used in clinical practice do not contain sufficient lipid ingredients required for the growth of Malassezia species. A recently developed medium, FastFung agar, is promising for culturing fastidious fungal species. METHODS: In this study, we compared FastFung agar and mDixon agar for culturing Malassezia species from nasolabial fold and retroauricular specimens of 83 healthy individuals and 187 and 57 patients with acne vulgaris and seborrheic dermatitis, respectively. RESULTS: Malassezia species were identified using conventional tests and matrix-assisted laser desorption/ionisation mass spectrometry. In total, 96 of 654 samples (14.6%) contained Malassezia species. The total isolation rate was significantly higher in patients with seborrheic dermatitis (40.4%) than in healthy volunteers (21.7%; p < .05), and the rate of M. furfur isolation was significantly higher for patients with acne vulgaris (13.9%) and seborrheic dermatitis (24.6%) than for healthy individuals (1.5%; p < .05). FastFung agar was superior to mDixon agar in M. furfur isolation (p = .004) but showed similar performance in the case of non-M. furfur species (p > .05). Among cultured Malassezia species, perfect agreement between mDixon agar and FastFung agar was found only for M. globosa (κ = 0.90). CONCLUSION: Our results indicate that FastFung agar favours the growth of Malassezia species and should be useful in clinical mycology laboratories.

Comparison of virulence factors and susceptibility profiles of Malassezia furfur from pityriasis versicolor patients and bloodstream infections of preterm infants.

In spite of the increasing medical interest in Malassezia yeasts, the virulence factors of Malassezia furfur causing bloodstream infections (BSI) were never investigated. Therefore, phospholipase (Pz), lipase (Lz), hemolysin (Hz), biofilm production, and in vitro antifungal susceptibility profiles were evaluated in M. furfur strains, isolated from both pityriasis versicolor (PV) patients (n = 18; Group 1) or from preterm infants BSI (n = 21; Group 2). All the test stains exhibited Pz activity, whereas 92.3% and 97.4% of strains exhibited Lz and Hz activities, respectively. Pz, Lz, and Hz activities were higher (i.e., lower values) within Group 1 strains (i.e., 0.48, 0.40, and 0.77) than those within Group 2 (i.e., 0.54, 0.54, and 0.81). The biofilm production was higher within Malassezia isolates from Group 2 (0.95 ± 0.3) than from Group 1 (0.72 ± 0.4). Itraconazole and posaconazole were the most active drugs against M. furfur, followed by amphotericin B and fluconazole. The minimum inhibitory concentrations (MIC) values varied according to the origin of M. furfur strains being statistically lower in M. furfur from Group 1 than from Group 2. This study suggests that M. furfur strains produce hydrolytic enzymes and biofilm when causing PV and BSI. Data show that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur BSI, whereas lipase and hemolytic activities might display a synergic role in skin infection.

There is no information on the virulence factors of M. furfur involved in invasive infections. Our data suggest that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur blood-stream infections.

Unveiling the structure of GPI-anchored protein of Malassezia globosa and its pathogenic role in pityriasis versicolor.

Glycosylphosphatidylinositols (GPI)-anchored proteins (GpiPs) are related to the cell wall biogenesis, adhesion, interactions, protease activity, mating, etc. These proteins have been identified in many organisms, including fungi such as Neurospora crassa, Candida albicans, Saccharomyces cerevisiae, and Fusarium graminearum. MGL-3153 gene of Malassezia globosa (M. globosa) encodes a protein which is homologous of the M. restricta, M. sympodialis, M. Pachydermatis, and U. maydis GpiPs. Real-time PCR assay showed that the expression of MGL_3153 gene was significantly up-regulated among M. globosa isolated from patients with pityriasis versicolor (PV) compared to a healthy individual, suggesting the contribution of this gene in the virulence of M. globosa. Accordingly, the sequence of this protein was analyzed by bioinformatics tools to evaluate the structure of that. The conservation analysis of MGL-3153 protein showed that the C-terminal region of this protein, which is responsible for GPI-anchor ligation, was highly conserved during evolution while the N-terminal region just conserved in Malassezia species. Moreover, the predicted tertiary structure of this protein by homology modeling showed that this protein almost has alpha helix structure and represented a stable structure during 150 ns of molecular dynamic simulation. Our results revealed that this protein potentially belongs to GPI-anchored proteins and may contribute to the virulence of M. globosa which warrants further investigations in this area.

Relationship between helicobacter pylori infection and pityriasis versicolor: can helicobacter pylori infection be a new etiologic factor for pityriasis versicolor?

Background/aim: H. pylori has been found to be related to certain dermatological diseases. However, there is no data as yet to propose an association between H. pylori and pityriasis versicolor. In this study, we aimed to evaluate the association between H. pylori and pityriasis versicolor. Materials and methods: This was a prospective study performed in the Gastroenterology and Dermatology and Venereology departments of the Health Sciences University, Ankara Training and Research Centre. A total of 57 consecutive patients (27 pityriasis versicolor, 30 telogen effluvium) were enrolled from the Department of Dermatology and Venereology. All patients were screened for H. pylori IgG and CagA. In addition, urea breath test was carried out to detect the existence of H. pylori infection. Results: There were significantly higher rates of H. pylori positivity, H. pylori IgG in serum in the pityriasis versicolor group compared to the telogen effluvium group (P < 0.05). In addition, the number of patients with dyspeptic complaints was higher in the pityriasis versicolor group than in the telogen effluvium group. The odds ratio for dyspepsia, H. pylori positivity, and H. pylori IgG were 2.48, 1.67, and 1.78, respectively. Conclusion: In this study, we found a statistically significant relationship between H. pylori infection and pityriasis versicolor. Therefore, H. pylori eradication could be considered in recurrent pityriasis versicolor patients with dyspepsia. New studies are required to clarify the effect of eradication treatment on the clinical course of pityriasis versicolor.

Comparative study between topically applied irradiated human amniotic membrane in combination with tea tree oil versus topical tioconazole in pityraisis versicolor treatment.

Pityriasis versicolor (PV) is a chronic skin disease caused by virulence activities of Malassezia, a genus of skin-associated yeasts. Traditionally, Tioconazole is used as a topical antifungal for curing PV. Previous investigations cited that human amniotic membrane (HAM), a placental tissue, has antimicrobial and anti-inflammatory activities and is useful as a dressing for healing skin lesions. Moreover, tea tree oil (TTO) has a potent antifungal efficacy. This clinical trial aims to achieve an alternative therapeutic treatment able to kill Malassezia and heal PV lesions using TTO-saturated HAM (TOSHAM), with little application times. This study subjected 120 patients with hypopigmented or hyperpigmented PV lesions; half patients were treated weekly with TOSHAM compared with the others who applying 1% Tioconazole cream daily as a traditional treatment. Microbiological evaluation of in vitro fungicidal activity of TOSHAM versus Tioconazole was carried out against Malassezia furfur culture. The clinical outcomes of this study proved the superior activity of TOSHAM to heal PV lesions than Tioconazole; this was in harmony with microbiological findings. This study approached a novel therapeutic treatment of PV with great outcomes by using TOSHAM.

Molecular epidemiology, in vitro susceptibility and exoenzyme screening of Malassezia clinical isolates.

Introduction. Malassezia folliculitis (MF) and pityriasis versicolor (PV) are common dermatoses caused by Malassezia species. Their molecular epidemiology, drug susceptibility and exoenzymes are rarely reported in China.Aim. To investigate the molecular epidemiology, drug susceptibility and enzymatic profile of Malassezia clinical isolates.Methodology. Malassezia strains were recovered from MF and PV patients and healthy subjects (HS) and identified by sequencing analysis. The minimum inhibitory concentrations (MICs) of nine antifungals (posaconazole, voriconazole, itraconazole, fluconazole, ketoconazole, miconazole, bifonazole, terbinafine and caspofungin) and tacrolimus, the interactions between three antifungals (itraconazole, ketoconazole and terbinafine) and tacrolimus, and the extracellular enzyme profile were evaluated using broth and checkerboard microdilution and the Api-Zym system, respectively.Results. Among 392 Malassezia isolates from 729 subjects (289 MF, 218 PV and 222 HS), Malassezia furfur and Malassezia globosa accounted for 67.86 and 18.88 %, respectively. M. furfur was the major species in MF and PV patients and HS. Among 60M. furfur and 50M. globosa strains, the MICs for itraconazole, posaconazole, voriconazole and ketoconazole were <1 µg ml-1. M. furfur was more susceptible to itraconazole, terbinafine and bifonazole but tolerant to miconazole compared with M. globosa (P<0.05). Synergistic effects between terbinafine and itraconazole or between tacrolimus and itraconazole, ketoconazole or terbinafine occurred in 6, 7, 6 and 9 out of 37 strains, respectively. Phosphatases, lipases and proteases were mainly secreted in 51 isolates.Conclusions. Itraconazole, posaconazole, voriconazole and ketoconazole are theagents against which there is greatest susceptibility. Synergistic effects between terbinafine and itraconazole or tacrolimas and antifungals may be irrelevant to clinical application. Overproduction of lipases could enhance the skin inhabitation of M. furfur.

Malassezia colonisation on a reconstructed human epidermis: Imaging studies.

BACKGROUND: Biofilm formation represents a major microbial virulence attribute especially at epithelial surfaces such as the skin. Malassezia biofilm formation at the skin surface has not yet been addressed. OBJECTIVE: The present study aimed to evaluate Malassezia colonisation pattern on a reconstructed human epidermis (RhE) by imaging techniques. METHODS: Malassezia clinical isolates were previously isolated from volunteers with pityriasis versicolor and seborrhoeic dermatitis. Yeast of two strains of M furfur and M sympodialis were inoculated onto the SkinEthic™ RHE. The tissues were processed for light microscopy, wide-field fluorescence microscopy and scanning electron microscopy. RESULTS: Colonisation of the RhE surface with aggregates of Malassezia yeast entrapped in a multilayer sheet with variable amount of extracellular matrix was unveiled by imaging techniques following 24, 48, 72 and 96 hours of incubation. Whenever yeast were suspended in RPMI medium supplemented with lipids, the biofilm substantially increased with a dense extracellular matrix in which the yeast cells were embedded. Slight differences were found in the biofilm architectural structure between the two tested species with an apparently higher entrapment and viscosity in M furfur biofilm. CONCLUSION: Skin isolates of M furfur and M sympodialis were capable of forming biofilm in vitro at the epidermal surface simulating in vivo conditions. Following 24 hours of incubation, without added lipids, rudimental matrix was barely visible, conversely to the reported at plastic surfaces. The amount of biofilm apparently increased progressively from 48 to 96 hours. A structural heterogeneity of biofilm between species was found.

Topical ketoconazole: a systematic review of current dermatological applications and future developments.

Introduction: Although labeling changes and market withdrawal have been implemented for oral ketoconazole (KTZ) due to serious adverse effects (AEs), topical KTZ is generally thought to be effective and safe for the treatment of superficial fungal infections. New dermatologic indications for the use of topical KTZ have arisen such as onychomycosis, blepharitis, and hair loss. This article aims to review the literature on topical KTZ's efficacy and AEs, as well as provide an overview on current insights regarding its mechanism of action and upcoming developments. Methods: A PubMed search was done to include randomized controlled trials (RCTs) focusing on the use of topical KTZ in human subjects. Results: Forty studies with 4566 patients were included in this review. Topical KTZ is clinically effective for the treatment of Malassezia-related conditions such as seborrheic dermatitis (SD) and pityriasis versicolor (PV) with a reported efficacy of 63-90% and 71-89%, respectively. Conclusions: Topical KTZ demonstrates high clinical efficacy for Malassezia-related conditions. More efficacious alternatives are now available for Tinea and Candida. Although topical KTZ is safe, clinicians should be aware that allergic contact dermatitis may occur. Further studies should be completed to investigate the use of topical KTZ for hair loss and inflammatory dermatoses.

Fungal infection in a post-renal transplant patient with Diaporthe.

Post-renal transplant fungal infections continue to be a major cause of mortality and morbidity. Universally reported fungi are Candida, especially Candida albicans, Cryptococcus, Aspergillus, Trichophyton rubrum and Pityriasis versicolor. Here, we report a case of infection caused by a rare fungus Diaporthe. It is an endophyte reported as plant pathogens and infrequently in humans and mammals. The patient was a renal transplant recipient on immunosuppressant. He had hypothyroidism and diagnosed with permanent pacemaker due to a complete heart block. The patient was treated with itraconazole (200 mg) successfully.

MALDI-TOF MS identification of Malassezia species isolated from patients with pityriasis versicolor at the seafarers' medical service in Dakar, Senegal.

Pityriasis versicolor (PV) is a superficial mycosis caused by yeast of the genus Malassezia. The most common isolated Malassezia species in PV lesions differ among M. furfur, M. globosa and M. sympodialis. We purpose to determine the distribution of Malassezia species in PV patients at the seafarers' medical service in Dakar, Senegal and to examine whether any association between identified Malassezia species and patients' profile. From May 2017 to August 2017, first a questionnaire was filled to get informative data before collection of skin scrapings taken from most scaly site using sterile scalpel blade and application of scotch® for direct examination (DE). At the laboratory, DE, culture and identification by MALDI-TOF MS were done. One hundred patients with PV - all men - were included with a mean age of 34 years. Among seafarers, 81% were sailors. Clinical prevalence of PV was highest in aged adults patients with ages of 31 to 60 years (56%). Seafarers with high level of education were less representative with only 2%. The mean duration of the PV was 26.83 months. 20% of subjects suffered lesions in more than one location. The chest was the most affected anatomical site. Furthermore, possible predisposing factors associated with PV were also detected. DE was positive in 95% but culture growth only in 46%. MALDI-TOF MS analysis of the positive cultures could be performed in 84.8% (39/46). Only M. furfur was identified in 100% (39/39). In definitive, M. furfur is the only causative agent of PV in Dakar.

MGL_3741 gene contributes to pathogenicity of Malassezia globosa in pityriasis versicolor.

Dihydroxyacid dehydratase (DHAD) is a key enzyme in biosynthetic pathway of isoleucine and valine. This pathway is absent in human but exists in various organisms such as fungi. Using RNA-seq analysis in this study, we identified MGL_3741gene which encodes DHAD protein in Malassezia globosa (M. globosa). Furthermore, we found that mentioned gene is homologous to the Ustilago maydis, Saccharomyces cerevisiae, Aspergillus flavus, and Aspergillus fumigatus ILV3P. For understanding the probable role of this gene in pathogenicity of M. globosa, we applied Real-time PCR to investigate the differentially expressed of the MGL_3741 gene in healthy and pathogenic states. Our results indicate a significant difference between two mentioned stats. These results revealed that ILV3-like gene in M. globosa can be related to the pathogenicity of this yeast.

Efficacy of modified Leeming-Notman media in a resazurin microtiter assay in the evaluation of in-vitro activity of fluconazole against Malassezia furfur ATCC 14521.

BACKGROUND: Malassezia furfur is lipodependent yeast like fungus that causes superficial mycoses such as pityriasis versicolor and dandruff. Nevertheless, there are no standard reference methods to perform susceptibility test of Malassezia species yet. AIMS: Therefore, in this study, we evaluated the optimized culture medium for growth of this lipophilic yeast using modified leeming-Notman agar and colorimetric resazurin microtiter assay to assess antimycotic activity of fluconazole against M. furfur. RESULTS: The result showed that these assays were more adjustable for M. furfur with reliable and reproducible MIC end-point, by confirming antimycotic activity of fluconazole with MIC of 2µg/ml. CONCLUSION: We conclude that this method is considered as the rapid and effective susceptibility testing of M. furfur with fluconazole antifungal activity.

Blad-containing oligomer: a novel fungicide used in crop protection as an alternative treatment for tinea pedis and tinea versicolor.

PURPOSE: The lack of novel antifungal drugs and the increasing incidence and severity of fungal infections are major concerns worldwide. Herein, we tested the activity of the Blad-containing oligomer (BCO), a new antifungal molecule already in use for agriculture, on Malassezia spp. and dermatophytes, the causal agents of human tinea versicolor and tinea pedis. Given the lack of a standard method for Malassezia susceptibility testing and the plethora of published methods, we also developed an improved method for this genus. METHODOLOGY: The efficacy of BCO was assessed in vitro and compared to that of the drugs currently utilized in the treatment of tinea versicolor (fluconazole and itraconazole) and tinea pedis (itraconazole and terbinafine). For dermatophytes, the standard microdilution broth-based method was used, with small adjustments, and several broth formulations and inocula sizes were tested to develop an improved susceptibility method for Malassezia spp. RESULTS: We successfully developed a microdilution broth-based method with considerable advantages over other available methods, and used it for all in vitro susceptibility tests of Malassezia spp. isolates. We report that, on a molar basis, BCO was more effective than fluconazole or itraconazole on most strains of Malassezia spp. isolated from clinical samples (n=29). By contrast, BCO was less effective than itraconazole or terbinafine on the common dermatophytes Trichophyton rubrum and Trichophyton interdigitale. CONCLUSION: These data place BCO as a promising drug for the treatment of Malassezia-associated skin diseases. Further in vivo studies are now required to ascertain its applicability in the clinical setting.