Molecular epidemiology of seborrheic dermatitis/dandruff associated Malassezia species from northern India.

Malassezia is a commensal that sometimes becomes pathogenic under the influence of diverse factors. Several species of Malassezia are difficult to culture, making traditional methods of identification challenging. The problem with molecular typing of Malassezia in association with seborrheic dermatitis/dandruff (SD/D) arises due to the unavailability of these fastidious yeast cultures. The aim of the study was to investigate the association between fluorescent amplified fragment length polymorphism (FAFLP) genotypes, disease state (SD/D), and the geographic distribution of M. globosa, M. restricta, and M. arunalokei. In total, 154 isolates representing M. globosa (n = 85), M. restricta (n = 55), and M. arunalokei (n = 14) from lesional/non-lesional areas of SD/D patients and healthy controls residing in the rural (n = 77) and urban (n = 77) areas of northern India were included. A strategy based on the FAFLP methodology was developed using two endonuclease enzymes (EcoRI and HindIII). M. globosa, M. restricta, and M. arunalokei formed 11, 3, and 2 FAFLP clusters, respectively. Disease-specific strains of M. restricta and M. arunalokei preferentially tend to cause SD/D. M. restricta and M. arunalokei showed less genetic variation. M.globosa showed higher genetic diversity. FAFLP clusters revealed the existence of geographically specific strains in M. restricta, M. arunalokei, and M. globosa. Our findings suggest that certain Malassezia strains are not only disease-specific but also geographically distinct.

The association of Malassezia with dandruff appears to be certain. Using the advanced technique, we determined that M. restricta and M. arunalokei are major species causing dandruff. There is also a difference in the specific molecular types affecting the rural and urban populations of India.

Invasive Pulmonary Infection of Malassezia Species in a Patient With Ulcerative Colitis: A Case Report.

Malassezia species are lipophilic yeasts recognized for causing skin manifestations, such as pityriasis versicolor. In addition, Malassezia can lead to invasive infection, mostly intravascular catheter-associated sepsis-related lipid-containing total parenteral nutrition in neonates and immunocompromised hosts. We experienced a case of invasive pulmonary Malassezia infection in a patient with refractory ulcerative colitis undergoing immunosuppressive treatment without lipid-containing total parenteral nutrition. Computed tomography (CT) images showed multiple lung nodules with CT halo signs. Finally, he underwent a partial right middle lobectomy and was diagnosed with invasive pulmonary malasseziosis through a genetic analysis. Multiple lung nodules on CT images may be found in invasive pulmonary malasseziosis in immunosuppressed patients with a central venous catheter.

Breast cancer colonization by Malassezia globosa accelerates tumor growth.

Malassezia globosa is a lipophilic basidiomycetous yeast that occurs abundantly in breast tumors and that may contribute to a shortened overall survival of breast cancer (BRAC) patients, suggesting that the yeast may participate in the carcinogenesis of BRAC. However, the mechanisms involved in the M. globosa-based acceleration of BRAC are unknown. Here, we show that M. globosa can colonize mammary tissue in 7,12-dimethylbenz[a] anthracene-induced mice. The abundance of M. globosa shortened the overall survival and increased the tumor incidence. Transcriptome data illustrated that IL-17A plays a key role in tumor growth due to M. globosa colonization, and tumor-associated macrophage infiltration was elevated during M. globosa colonization which triggers M2 polarization of macrophages via toll-like receptors 4/nuclear factor kappa-B (Nf-κB) signaling. Our results show that the expression of sphingosine kinase 1 (Sphk1) is increased in breast tumors after inoculation with M. globosa. Moreover, we discovered that Sphk1-specific small interfering RNA blocked the formation of lipid droplets, which can effectively alleviate the expression of the signal transducer and activator of the transcription 3 (STAT3)/Nf-κB pathway. Taken together, our results demonstrate that M. globosa could be a possible factor for the progression of BRAC. The mechanisms by which M. globosa promotes BRAC development involve the IL-17A/macrophage axis. Meanwhile, Sphk1 overexpression was induced by M. globosa infection, which also promoted the proliferation of MCF-7 cells.IMPORTANCELiterature has suggested that Malassezia globosa is associated with breast tumors; however, this association has not been confirmed. Here, we found that M. globosa colonizes in breast fat pads leading to tumor growth. As a lipophilic yeast, the expression of sphingosine kinase 1 (Sphk1) was upregulated to promote tumor growth after M. globosa colonization. Moreover, the IL-17A/macrophages axis plays a key role in mechanisms involved in the M. globosa-induced breast cancer acceleration from the tumor immune microenvironment perspective.

Malassezia globosa Induces Differentiation of Pathogenic Th17 Cells by Inducing IL-23 Secretion by Keratinocytes.

Malassezia, the most abundant fungal commensal on the mammalian skin, has been linked to several inflammatory skin diseases such as atopic dermatitis, seborrheic dermatitis and psoriasis. This study reveals that epicutaneous application with Malassezia globosa (M. globosa) triggers skin inflammation in mice. RNA-sequencing of the resulting mouse lesions indicates activation of Interleukin-17 (IL-17) signaling and T helper 17 (Th17) cells differentiation pathways by M. globosa. Furthermore, our findings demonstrate a significant upregulation of IL-23, IL-23R, IL-17A, and IL-22 expressions, along with an increase in the proportion of Th17 and pathogenic Th17 cells in mouse skin exposed to M. globosa. In vitro experiments illustrate that M. globosa prompts human primary keratinocytes to secrete IL-23 via TLR2/MyD88/NF-κB signaling. This IL-23 secretion by keratinocytes is shown to be adequate for inducing the differentiation of pathogenic Th17 cells in the skin. Overall, these results underscore the significant role of Malassezia in exacerbating skin inflammation by stimulating IL-23 secretion by keratinocytes and promoting the differentiation of pathogenic Th17 cells.

Evaluation of an in-house pan-Malassezia quantitative PCR in human clinical samples.

Althought Malassezia spp. have been involved in various pathologies, they are an integral part of the cutaneous, gut, oral, ears, nose and throat (ENT) mycobiota. Since Malassezia are difficult to grow in culture, unexhaustive molecular biology methods have been developed to detect them. The aim of the study was to evaluate an in-house pan-Malassezia quantitative polymerase chain reaction (panM-qPCR) on various clinical human samples and determine Malassezia burden in various human mycobiota. The panM-qPCR was designed to target the repeated 28S rDNA gene from all Malassezia species. We used the assay to quantify the Malassezia burden on 361 samples from 161 subjects (80 skin swabs from 10 healthy volunteers (HV), 13 samples from 2 seborrheic dermatitis patients (SD), 90 skin samples from 19 burned patients, 119 stool samples from 89 immunocompromised patients, 59 ENT samples from 41 patients). For HV, the amount of Malassezia was different according to the swabbed areas. Quantification cycle (Cq) in SD is lower than in HV. In burned patients, Cq was significantly lower compared to HV. In stool samples, 6.7% were positive for Malassezia spp. with a high Cq. For the ENT area, a higher proportion of positive specimens were detected in ear samples than in nose samples. Our findings emphasized the importance of qPCR, confirming elevated Malassezia spp. levels on individuals' faces and scalps, increased burden in SD patients and in severely burnt patients than in HV. The pan-MqPCR appears to be a promising tool for studying Malassezia in various human mycobiota.

Malassezia species are ubiquitous members of various human microbiomes, including cutaneous and mucosal sites. While these fungi have been implicated in several pathologies, their presence as commensals complicates their study, especially due to difficulties in culturing them in vitro. This has necessitated the development of molecular techniques to detect and quantify Malassezia species directly from clinical samples. In this study, we report on the development and application of an in-house pan-Malassezia quantitative PCR (panM-qPCR) assay. This assay targets the conserved 28S rDNA gene across all known Malassezia species, allowing for a broad-spectrum detection approach. We applied this panM-qPCR to a diverse set of clinical samples, totaling 361 specimens from 161 subjects, encompassing healthy individuals, patients with seborrheic dermatitis, burn victims, and immunocompromised individuals. Our results indicate variable Malassezia loads on different skin sites of healthy volunteers, with significantly lower quantification cycle (Cq) values observed in seborrheic dermatitis patients, suggesting an increased fungal burden. Burn patients also showed a marked increase in Malassezia spp. levels compared to healthy individuals. Stool samples demonstrated a low prevalence (6.7%) of Malassezia spp., but with high Cq values when present. Notably, ear samples revealed a higher positivity rate compared to nasal samples. The findings highlight the practicality and sensitivity of qPCR for elucidating the Malassezia burden across various human samples. This molecular approach confirms the differential colonization of Malassezia spp. in different clinical contexts. The panM-qPCR offers a promising approach for comprehensive mycobiota research, particularly in conditions where culture-based methods fall short.

Anti-dandruff effects of butterfly pea flowers (Clitoria ternatea)-based shampoo: A pretest-posttest control study.

Butterfly pea flower (Clitoria ternatea) may serve as an alternative anti-dandruff treatment; however, its effects on Malassezia spp. remain unexplored. The aim of this study was to explore the effects of C. ternatea as an herbal-based anti-dandruff treatment on Malassezia spp. DNA expression, plakoglobin levels, IL-8 levels, sebum levels, dandruff severity scores, adverse effects, and patient satisfaction. An experimental study with a pretest-posttest control design was conducted at the Outpatient Clinic of Dermatology and Venereology, Arifin Achmad Hospital, Pekanbaru, Indonesia, from November 2023 to January 2024. The flower of C. ternatea was used to formulate the shampoo. The study involved 70 female patients aged 18-25 with dandruff, who were divided into two groups: (a) experimental group using 20% C. ternatea shampoo and (b) control group using 2% ketoconazole shampoo. The present study found that 2% ketoconazole shampoo significantly reduced Malassezia spp. DNA expression compared to 20% C. ternatea shampooo (Clitoria ternatea: ΔCq=1.76±3.18; ketoconazole: ΔCq=3.77±2.90; p=0.008). No significant difference was observed in plakoglobin levels (C. ternatea: ΔCq=1.98±3.63; ketoconazole: ΔCq=2.50±2.36; p=0.427) or IL-8 levels (C. ternatea: ΔCq=3.46±4.00; ketoconazole: ΔCq=4.16 ± 3.62; p=0.459). C. ternatea significantly reduced sebum levels more than ketoconazole (C. ternatea: 1.16±0.98%; ketoconazole: 0.22±0.38%; p<0.001). Dandruff scores and patient satisfaction were similar for both shampoos (p=0.115 and p=0.336, respectively). Adverse effects were more common in the 2% ketoconazole shampoo group, affecting 21.2% of the patients. In conclusion, 2% ketoconazole shampoo is more effective in reducing Malassezia spp. DNA expression, while 20% C. ternatea shampoo offers better sebum control. Both shampoos are similarly effective in ameliorating dandruff severity and are well-tolerated, with fewer adverse effects reported for C. ternatea.

Paenibacillus exopolysaccharide alleviates Malassezia-induced skin damage: Enhancing skin barrier function, regulating immune responses, and modulating microbiota.

Numerous studies have established a strong association between Malassezia and various skin disorders, including atopic dermatitis. Finding appropriate methods or medications to alleviate Malassezia-induced skin damage is of notable public interest. This study aimed to evaluate the therapeutic effect of the exopolysaccharide EPS1, produced by Paenibacillus polymyxa, on Malassezia restricta-induced skin damage. In vitro assays indicated that EPS1 reduced the expression of pro-inflammatory cytokine genes in TNF-α-induced HaCaT cells. In a murine model, EPS1 was found to mitigate clinical symptoms, reduce epidermal thickness and mast cell infiltration, improve skin barrier function, decrease pro-inflammatory cytokine levels associated with type 17 inflammation, enhance Tregs in the spleen, upregulate the transcription of Treg-related genes in skin lesions, and modulate the skin microbiota. This study is the first to report the alleviating effect of Paenibacillus exopolysaccharide on Malassezia-induced skin inflammation and its impact on the skin microbiota. These findings support the potential of Paenibacillus exopolysaccharides as consumer products and therapeutic agents for managing Malassezia-induced skin damage by improving skin barrier function, modulating immune responses, and influencing skin microbiota.

Characteristics of Malassezia furfur at various pH and effects of Malassezia lipids on skin cells.

Malassezia species are commensal and opportunistic fungi found in human skin. All Malassezia species lack fatty acid synthesis genes and survive by utilizing several lipases to degrade and absorb fatty acids from external lipid sources, but little research has been done on their optimal active pH and temperature. Our skin protects itself from external stimuli and maintains homeostasis, involving bacteria and fungi such as Malassezia species that inhabit our skin. Hence, dysbiosis in the skin microbiome can lead to various skin diseases. The skin's pH is slightly acidic compared to neutral, and changes in pH can affect the metabolism of Malassezia species. We used keratinocyte cell lines to determine the effect of lipids bio-converted by Malassezia furfur, Malassezia japonica, and Malassezia yamatoensis under pH conditions similar to those of healthy skin. Lipids bio-converted from Malassezia species were associated with the regulation of transcripts related to inflammation, moisturizing, and promoting elasticity. Therefore, to determine the effect of pH on lipid metabolism in M. furfur, which is associated with seborrheic dermatitis, changes in biomass, lipid content, and fatty acid composition were determined. The results showed that pH 7 resulted in low growth and reduced lipid content, which had a negative impact on skin health. Given that bio-converted Malassezia-derived lipids show positive effects at the slightly acidic pH typical of healthy skin, it is important to study their effects on skin cells under various pH conditions. KEY POINTS: • pH 6, Malassezia spp. bio-converted lipid have a positive effect on skin cells • Malassezia spp. have different lipid, fatty acid, and growth depending on pH • Malassezia spp. can play a beneficial role by secreting lipids to the outside.

Enhanced visualization of nuclear staining and cell cycle analysis for the human commensal Malassezia.

Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.

Experimental and computational insights of Albizia amara phytoconstituents targeting anthranilate phosphoribosyltransferase from Malassezia globosa.

The fungus Malassezia globosa is often responsible for superficial mycoses posing significant treatment challenges because of the unfavourable side effects of available antifungal drugs. To reduce potential hazards to the host and overcome these hurdles, new therapeutic medicines must be developed that selectively target enzymes unique to the pathogen. This study focuses on the enzyme anthranilate phosphoribosyltransferase (AnPRT), which is vital to M. globosa's tryptophan production pathway. To learn more about the function of the AnPRT enzyme, we modeled, validated, and simulated its structure. Moreover, many bioactive components were found in different extracts from the plant Albizia amara after phytochemical screening. Interestingly, at doses ranging from 500 to 2000 µg/ml, the chloroform extract showed significant antifungal activity, with inhibition zones measured between 11.0 ± 0.0 and 25.6 ± 0.6 mm. According to molecular docking analyses, the compounds from the active extract, particularly 2-tert-Butyl-4-isopropyl-5-methylphenol, interacted with the AnPRT enzyme's critical residues, ARG 205 and PHE 214, with an effective binding energy of -4.9 kcal/mol. The extract's revealed component satisfies the requirements for drug-likeness and shows promise as a strong antifungal agent against infections caused by M. globosa. These findings imply that using plant-derived chemicals to target the AnPRT enzyme is a viable path for the creation of innovative antifungal treatments.

Concurrent genotyping and expression of NLRP3 inflammasome in pityriasis versicolor patient's skin lesions.

The goal of this study is to investigate the impact of the rs35829419 SNP on the serum level of NLRP3, and to assess the relationship between NLRP3 and its SNP and vulnerability to Pityriasis versicolor. Pityriasis versicolor (PV) is one of the most frequent skin conditions linked to skin pigmentation changes. Malassezia plays a key role in the pathogenesis of PV. A case-control study, 50 patients with pityriasis versicolor and 44 healthy controls. Real-time PCR was used to genotype NLRP3 (rs35829419) and ELISA assay of NLRP3 levels in tissue samples. There was a significantly higher median NLPR3 levels in PV patients than controls. A significant predominance of A allele of Q 705 K was in patients than controls. The risk of having the disease in the presence of A allele is nearly 10 times than having C allele. In PV patients, there was a significant relationship between NLPR3 levels and Q 705 K genotypes with higher NLPR3 levels in AA genotype. A potential correlation between PV and the Q705K polymorphism, pointing to evidence of NLRP3 alteration in PV patients. The NLRP3 inflammasome may be an appropriate therapeutic target for Malassezia-associated skin disorders.

Malassezia responds to environmental pH signals through the conserved Rim/Pal pathway.

During mammalian colonization and infection, microorganisms must be able to rapidly sense and adapt to changing environmental conditions including alterations in extracellular pH. The fungus-specific Rim/Pal signaling pathway is one process that supports microbial adaptation to alkaline pH. This cascading series of interacting proteins terminates in the proteolytic activation of the highly conserved Rim101/PacC protein, a transcription factor that mediates microbial responses that favor survival in neutral/alkaline pH growth conditions, including many mammalian tissues. We identified the putative Rim pathway proteins Rim101 and Rra1 in the human skin colonizing fungus Malassezia sympodialis. Gene deletion by transconjugation and homologous recombination revealed that Rim101 and Rra1 are required for M. sympodialis growth at higher pH. In addition, comparative transcriptional analysis of the mutant strains compared to wild-type suggested mechanisms for fungal adaptation to alkaline conditions. These pH-sensing signaling proteins are required for optimal growth in a murine model of atopic dermatitis, a pathological condition associated with increased skin pH. Together, these data elucidate both conserved and phylum-specific features of microbial adaptation to extracellular stresses.IMPORTANCEThe ability to adapt to host pH has been previously associated with microbial virulence in several pathogenic fungal species. Here we demonstrate that a fungal-specific alkaline response pathway is conserved in the human skin commensal fungus Malassezia sympodialis (Ms). This pathway is characterized by the pH-dependent activation of the Rim101/PacC transcription factor that controls cell surface adaptations to changing environmental conditions. By disrupting genes encoding two predicted components of this pathway, we demonstrated that the Rim/Pal pathway is conserved in this fungal species as a facilitator of alkaline pH growth. Moreover, targeted gene mutation and comparative transcriptional analysis support the role of the Ms Rra1 protein as a cell surface pH sensor conserved within the basidiomycete fungi, a group including plant and human pathogens. Using an animal model of atopic dermatitis, we demonstrate the importance of Ms Rim/Pal signaling in this common inflammatory condition characterized by increased skin pH.

Cold atmospheric plasma therapy for Malassezia folliculitis: Laboratory investigations and a randomized clinical trial.

BACKGROUND: Current treatment options for Malassezia folliculitis (MF) are limited. Recent research has demonstrated the inhibitory effect of cold atmospheric plasma (CAP) on the growth of Malassezia pachydermatis in vitro, suggesting CAP as a potential therapeutic approach for managing MF. OBJECTIVES: The objective of our study is to assess the in vitro antifungal susceptibility of Malassezia yeasts to CAP. Additionally, we aim to evaluate the efficacy and tolerability of CAP in treating patients with MF. METHODS: We initially studied the antifungal effect of CAP on planktonic and biofilm forms of Malassezia yeasts, using well-established techniques such as zone of inhibition, transmission electron microscopy, colony count assay and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt assay. Subsequently, a randomized (1:1 ratio), active comparator-controlled, observer-blind study was conducted comparing daily CAP therapy versus itraconazole 200 mg/day for 2 weeks in 50 patients with MF. Efficacy outcomes were measured by success rate, negative microscopy rate and changes in Dermatology Life Quality Index (DLQI) and Global Aesthetic Improvement Scale (GAIS) scores. Safety was assessed by monitoring adverse events (AEs) and local tolerability. RESULTS: In laboratory investigations, CAP time-dependently inhibited the growth of Malassezia yeasts in both planktonic and biofilm forms. Forty-nine patients completed the clinical study. At week 2, success was achieved by 40.0% of subjects in the CAP group versus 58.3% in the itraconazole group (p = 0.199). The negative direct microscopy rates of follicular samples were 56.0% in the CAP group versus 66.7% in the itraconazole group (p = 0.444). No significant differences were found in the proportion of subjects achieving DLQI scores of 0/1 (p = 0.456) or in the GAIS responder rates (p = 0.588) between the two groups. Three patients in the CAP group and one patient in the itraconazole group reported mild AEs. CONCLUSION: CAP demonstrated significant antifungal activity against Malassezia yeasts in vitro and exhibited comparable efficacy to itraconazole in treating MF patients. Without the associated adverse effects of oral antifungal drugs, CAP can be considered a promising and safe treatment modality for MF.

Next generation mycological diagnosis: Artificial intelligence-based classifier of the presence of Malassezia yeasts in tape strip samples.

BACKGROUND: Malassezia yeasts are almost universally present on human skin worldwide. While they can cause diseases such as pityriasis versicolor, their implication in skin homeostasis and pathophysiology of other dermatoses is still unclear. Their analysis using native microscopy of skin tape strips is operator dependent and requires skill, training and significant amounts of hands-on time. OBJECTIVES AND METHODS: To standardise and improve the speed and quality of diagnosis of Malassezia in skin tape strip samples, we sought to create an artificial intelligence-based algorithm for this image classification task. Three algorithms, each using different internal architectures, were trained and validated on a manually annotated dataset of 1113 images from 22 samples. RESULTS: The Vision Transformer-based algorithm performed the best with a validation accuracy of 94%, sensitivity of 94.0% and specificity of 93.5%. Visualisations providing insight into the reasoning of the algorithm were presented and discussed. CONCLUSION: Our image classifier achieved very good performance in the diagnosis of the presence of Malassezia yeasts in tape strip samples of human skin and can therefore improve the speed and quality of, and access to this diagnostic test. By expanding data sources and explainability, the algorithm could also provide teaching points for more novice operators in future.

In vitro effects of N-acetylcysteine in combination with antifungal agents against Malassezia pachydermatis isolated from canine otitis externa.

BACKGROUND: Many clinicians prescribe antifungal agents to treat canine otitis externa (OE). However, studies evaluating the antifungal effects of N-acetylcysteine (NAC) and its combinations are limited. HYPOTHESIS/OBJECTIVES: The aim of this study was to evaluate the antifungal effects of NAC alone and in combination with other antifungal agents against Malassezia pachydermatis isolated from canine OE. MATERIALS AND METHODS: M. pachydermatis samples were collected from 13 dogs with OE. The final concentration of the inoculum suspensions of M. pachydermatis was 1-5 × 106 colony forming units/mL. The concentrations of the test compounds ketoconazole (KTZ), terbinafine (TER), nystatin (NYS) and NAC were 0.02-300 µg/mL, 0.04-80 µg/mL, 0.16-40 µg/mL and 1.25-20 mg/mL, respectively. The minimum inhibitory concentration (MIC) was measured to evaluate the susceptibility of the M. pachydermatis to KTZ, TER, NYS and NAC. The checkerboard testing method and fractional inhibitory concentration index were used to evaluate the effect of NAC in combination with KTZ, TER and NYS against M. pachydermatis. RESULTS: The MIC90 values of M. pachydermatis were 4.6875-9.375 µg/mL, 1.25 µg/mL, 5-10 µg/mL and 10 mg/mL for KTZ, TER, NYS and NAC, respectively. The synergistic effects of KTZ, TER and NYS with NAC were identified in 0/13, 2/13 and 0/13 isolates, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: NAC had an antifungal effect against M. pachydermatis but did not exert synergistic effects when used with KTZ, TER and NYS. Thus, the use of NAC alone as a topical solution could be considered an effective treatment option for canine OE involving M. pachydermatis.

Fungal Head and Neck Dermatitis: Current Understanding and Management.

Head and neck dermatitis (HND) is a form of atopic dermatitis (AD) that affects the seborrheic areas of the body and causes greater quality of life detriments than other types of AD. HND can be challenging to treat since first-line topical therapies may be ineffective or intolerable for long-term use on areas affected by HND while dupilumab may cause dupilumab-associated HND (DAHND). Current evidence implicates fungi, particularly Malassezia spp., in the pathogenesis of HND. Penetration of fungal antigens through the defective AD skin barrier activates the innate and adaptive immune systems to cause cutaneous inflammation via the T helper (Th)17 and/or Th2 axes. Malassezia sensitization may distinguish HND from other forms of AD. Multiple double-blind, placebo-controlled trials have shown antifungals to benefit HND, yet the persistence of symptom relief with sustained use remains unclear. Oral antifungals appear more effective than topical antifungals but may be harmful with long-term use. DAHND may also be fungal-mediated given improvement with antifungals and evidence of an overactive immune response against Malassezia in these patients. Janus kinase inhibitors are effective for HND, including DAHND, but may cause significant side effects when administered systemically. OX40/OX40L inhibitors and tralokinumab may be promising options for HND on the horizon. Demographic and environmental factors influence the host mycobiome and should be considered in future precision-medicine approaches as microbiome composition and diversity are linked to severity of HND.

Malassezia restricta as an unexpected cause of infectious osteomyelitis diagnosed by metagenomic sequencing: a case report and literature review.

BACKGROUND: Malassezia restricta, a lipophilic and lipodependent yeast belonging to the basidiomycetes group, is an opportunistic fungal pathogen associated with various skin diseases, including seborrheic dermatitis and dandruff. Typically, Malassezia infection in neonates manifests as fungemia or hematogenous dissemination to the bone or lungs. However, vertebral osteomyelitis caused by these fungi is rarely reported owing to non-specific clinical presentations and laboratory/imaging findings. The Pathogen Metagenomics Sequencing (PMseq) technique enables direct high-throughput sequencing of infected specimens, facilitating the rapid and accurate detection of all microorganisms in clinical samples through comprehensive reports. CASE PRESENTATION: A 52-year-old male was admitted to our hospital on July 20, 2022 with a 3-month history of ambulatory difficulties and localized low back pain. Magnetic Resonance Imaging (MRI) examination of the spinal column revealed irregular bone destruction affecting the L2, L3, and L5 vertebral bodies. Additionally, low T1 and high T2 intensity lesions were observed at the intervertebral discs between L3 and L5. The presumptive diagnosis of tuberculous spondylitis was made based on the imaging findings, despite negative results in all mycobacterium tests. However, the patient exhibited no improvement after receiving regular anti-tuberculosis treatment for 3 months. Subsequent MRI revealed an expansive abnormal signal within the vertebral body, leading to progressive bone destruction. The absence of spinal tuberculosis or other infective microorganisms was confirmed through culture from blood and pathological tissue from the L4 vertebral body. Subsequently, PMseq was performed on the specimens, revealing M. restricta as the predominant pathogen with the highest relative abundance value. The pathological examination revealed the presence of fungal mycelium in the L4 vertebral body, with positive findings on periodic Schiff-methenamine and periodic acid-Schiff staining. The anti-tuberculosis treatment was discontinued, and an antifungal combination of fluconazole and voriconazole was administered. All symptoms were resolved after 7 consecutive months of treatment, and the patient was able to ambulate autonomously. Vertebral lesions were reduced on MRI during the 13-month follow-up. CONCLUSIONS: M. restricta is not a commonly recognized pathogen associated with infectious vertebral osteomyelitis. However, PMseq can aid in diagnosis, timely treatment, and decision making for some non-specific infectious diseases.

Action of mycocins produced by Wickerhamomyces anomalus on Malassezia pachydermatis isolated from the ear canal of dogs.

This study aims to examine the effectiveness of mycocins produced by Wickerhamomyces anomalus in inhibiting Malassezia pachydermatis, a yeast commonly found in the ear canal of dogs. M. pachydermatis has a zoophilic origin and can be found in mammals, and frequently in dogs, where it mainly colonizes the ear canal region and the skin, leading to lesions that are difficult to treat. The antimicrobial mechanism was evaluated using dilutions of supernatant with enzymatic activity, which may include ß-glucanases, glycoproteins known to act on microorganism cell walls. However, it is important to note that this supernatant may contain other compounds as well. ß-glucanases in the mycocins supernatant were found at a concentration of 0.8 U/mg. The susceptibility of M. pachydermatis isolates was tested using the microdilution method. The isolates suffered 100% inhibition when tested with the culture supernatant containing mycocins. In the proteinases production test, 44% of the isolates tested were strong proteinases producers. Subsequently all these isolates suffered inhibition of their activity when tested in research medium containing mycocins supernatant at a subinhibitory concentration of ß-glucanases. This shows that mycocins can inhibit the production of proteinases, a virulence factor of M. pachydermatis. The viability test showed the antifungal action of mycocins in inhibiting the viability of M. pachydermatis cells after a period of 8  hours of contact. These results support the antimicrobial potential of mycocins and their promise as a therapeutic option.

Extracellular phospholipase production by Malassezia pachydermatis strains and its inhibition by selected antimycotics and plant essential oil components.

Extracellular phospholipase (EPL) plays an important role in the pathogenesis of the yeast Malassezia pachydermatis. Currently, the attention of researchers is focused on studying the virulence factors involved in this process and searching solutions to reduce their activity. One of the options is the use of natural remedies as anti-virulence agents. This study is aimed at investigating the production of extracellular phospholipase in M. pachydermatis strains (18 samples) and followed by the time-dependent inhibitory effect of selected azole antifungals (itraconazole, posaconazole and voriconazole) and plant essential oil components (terpinen-4-ol, thymol, carvacrol, eugenol and geraniol), evaluated by Egg Yolk Agar plate method. Almost all strains (17 isolates, (94.4%) were found to be intense EPL producers. A significant, time-dependent inhibition of EPL was noted after 1-, 3- and 6-h exposure of Malassezia cells to itraconazole (26.4%, 47.2% and 50.9%, respectively) compared to exposure to posaconazole (26.4%, 28.3% and 28.3%, respectively) and voriconazole (18.8%, 20.8% and 35.8%, respectively). After one-hour exposure to plant essential oil components, the best inhibitory effect was recorded for eugenol (62.3%), followed by terpinen-4-ol and thymol (56.6%), geraniol (41.5%) and carvacrol (26.4%). A 3-h exposure revealed that thymol retained the best inhibitory effect (88.7%) on EPL production, followed by carvacrol (73.6%), eugenol (56.6%), terpinen-4-ol (52.8%) and geraniol (49.1%). After 6-h exposure, no growth of M. pachydermatis strains exposed to carvacrol was observed, and the inhibitory efficiency for the other tested essential oil (EO) components achieved 88.7%. The obtained results indicate the promising efficacy of plant essential oils components in the inhibition of virulence factors such as EPL production.

Exploring the antibiofilm efficacy of cinnamaldehyde against Malassezia globosa associated pityriasis versicolor.

BACKGROUND: Malassezia globosa is a commensal basidiomycetous yeast occurring on the skin that causes pityriasis versicolor (PV) and seborrheic dermatitis, but that has also been implicated in other dermatoses. Cinnamaldehyde (CM) has antibacterial, antioxidant, and anti-inflammatory activities, but the effect of CM on M. globosa-infected PV has not been clarified. PURPOSE: The study aimed to investigate the possible antifungal and antibiofilm activities of CM against M. globosa-infected PV in vivo and in vitro. METHODS: The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of CM against M. globosa. The crystal violet staining assay and XTT assay were used to investigate the inhibition of CM on biofilm formation and the eradication of mature biofilms. The visualizations of the biofilm and cell distribution in the biofilm matrix were performed with a scanning electron microscope and confocal laser scanning microscope. The kits of antioxidant kinase were used to determine the activities of oxidative stress markers in M. globosa-stimulated HaCaT cells. Western blot assays were used to evaluate the role of TLR2/NF-κB in vitro. Furthermore, the protective effect of CM was assessed in M. globosa-associated PV mice. The expressions of inflammatory cytokines and apoptosis were screened using ELISA assays. The expressions of interleukin-6 and tumor necrosis factor-α were measured by an immunohistochemistry method in vivo. RESULTS: Our results showed that the MIC of CM against planktonic cells of M. globosa was 4 µg/ml and treatment with 20 × MIC CM eradicated mature biofilms of M. globosa. In vitro, after CM treatment the levels of oxidative stress indicators (i.e., superoxide dismutase, catalase, glutathione) significantly increased, while the levels of malondialdehyde decreased. In addition, the expression of TLR2/NF-κB in HaCaT cells was significantly reduced after CM treatment. On the other hand, an in vivo therapeutic effect of CM was assessed against M. globosa-infected mice. The fungal load on the skin decreased after treatment with CM compared to the M. globosa-infected group. In addition, the uninfected animals showed a normal skin structure, whereas, the M. globosa-infected mice showed extensive infiltration of neutrophils in skin tissues that improved after treatment with CM. Meanwhile, the levels of inflammatory and apoptotic factors improved after CM treatment. CONCLUSION: Our results showed that CM inhibits the biofilm formation of M. globosa and eradicates mature biofilms of M. globosa. Treatment with CM significantly decreased oxidative stress, apoptosis, and inflammatory markers in the skin tissue and HaCaT cells. Hence, this study suggests that CM is a good candidate therapeutic agent against M. globosa-induced PV infections because of its antifungal, antibiofilm, and anti-inflammatory properties.