Neutrophil extracellular traps (NETs) and Th-2 dominant immune responses in chronic granulomatous chromobalstomycosis.

Chromoblastomycosis (CBM), a chronic, granulomatous, suppurative mycosis of the skin and subcutaneous tissue, is caused by several dematiaceous fungi. The formation of granulomas, tissue proliferation, and fibrosis in response to these pathogenic fungi is believed to be intricately linked to host immunity. To understand this complex interaction, we conducted a comprehensive analysis of immune cell infiltrates, neutrophil extracellular traps (NETs) formation, and the fibrosis mechanism in 20 CBM lesion biopsies using immunohistochemical and immunofluorescence staining methods. The results revealed a significant infiltration of mixed inflammatory cells in CBM granulomas, prominently featuring a substantial presence of Th2 cells and M2 macrophages. These cells appeared to contribute to the production of collagen I and III in the late fibrosis mechanism, as well as NETs formation. The abundance of Th2 cytokines may act as a factor promoting the bias of macrophage differentiation toward M2, which hinders efficient fungal clearance while accelerates the proliferation of fibrous tissue. Furthermore, the expression of IL-17 was noted to recruit neutrophils, facilitating subsequent NETs formation within CBM granulomas to impede the spread of sclerotic cells. Understanding of these immune mechanisms holds promise for identifying therapeutic targets for managing chronic granulomatous CBM.

Successful management of chromoblastomycosis utilizing conventional antifungal agents and imiquimod therapy.

Chromoblastomycosis (CBM), a chronic fungal infection affecting the skin and subcutaneous tissues, is predominantly caused by dematiaceous fungi in tropical and subtropical areas. Characteristically, CBM presents as plaques and nodules, often leading to scarring post-healing. Besides traditional diagnostic methods such as fungal microscopy, culture, and histopathology, dermatoscopy and reflectance confocal microscopy can aid in diagnosis. The treatment of CBM is an extended and protracted process. Imiquimod, acting as an immune response modifier, boosts the host's immune response against CBM, and controls scar hyperplasia, thereby reducing the treatment duration. We present a case of CBM in Guangdong with characteristic reflectance confocal microscopy manifestations, effectively managed through a combination of itraconazole, terbinafine, and imiquimod, shedding light on novel strategies for managing this challenging condition.

NLRP3 inflammasome activation contributes to fungal clearance and lung injury during Talaromyces marneffei infection.

The increased levels of IL-1ß and IL-18 cytokines have been associated with the severity of sepsis and outcomes of patients infected with Talaromyces marneffei. Previous studies have suggested that NLRP3 plays an important role in caspase-1 activated secretion of IL-1ß and IL-18 in fungal-infected macrophages. In the present study, the role of the NLRP3 inflammasome in talaromycosis is investigated in an in vitro assay and in vivo with a mice systemic infection model. We found that the NLRP3 inflammasome pathway in infected mice is activated along with increased production of IL-1ß. Such an activation of the NLRP3 inflammasome is also observed in either mice or human macrophages challenged with T. marneffei conidia. Our results indicate that IL-1ß release by infected macrophages is NLRP3 inflammasome-dependent and NLRP3 contributes to death of mice at the early stage of pulmonary infection. Moreover, a greater number of MPO-positive cells are found in the lungs of infected Nlrp3-/- mice and WT mice with reduced LDH levels, especially at the last stage of infection. Therefore, we conclude that the NLRP3 Inflammasome activation is important for fungal clearance, neutrophil recruitment and lung injury during T. marneffei Infection.

Autophagy benefits the in vitro and in vivo clearance of Talaromyces marneffei.

Talaromycosis, namely Talaromyces marneffei infection, is increasing gradually and has a high mortality rate even under antifungal therapy. Although autophagy acts differently on different pathogens, it is a promising therapeutic strategy. However, information on autophagy in macrophages and animals upon infection by T. marneffei is still limited. Therefore, several models were employed here to investigate the role of autophagy in host defense against T. marneffei, including RAW264.7 macrophages as in vitro models, different types of Caenorhabditis elegans and BALB/c mice as in vivo models. We applied the clinical T. marneffei isolate SUMS0152 in this study. T. marneffei-infected macrophages exhibit increased formation of autophagosomes. Further, macrophage autophagy promoted by rapamycin or Earle's balanced salt solution (EBSS) inhibited the viability of intracellular T. marneffei. In vivo, compared with uninfected Caenorhabditis elegans, the wild-type nematodes upregulated the expression of the autophagy-related gene lgg-1 and atg-18, and nematodes carrying GFP reporter were induced to form autophagosomes (GFP::LGG-1) after T. marneffei infection. Furthermore, the knockdown of lgg-1 significantly reduced the survival rate of T. marneffei-infected nematodes. Likewise, the autophagy activator rapamycin reduced the fungal burden and suppressed lung inflammation in a mouse model of infection. In conclusion, autophagy is essential for host defense against T. marneffei in vitro and in vivo. Therefore, autophagy may be an attractive target for developing new therapeutics to treat talaromycosis.

The relationship between the preference of mating type (MAT) and source in the opportunistic pathogen Talaromyces marneffei.

Genome-wide comparisons have shown Talaromyces marneffei possessed a stable mating type locus in its meiosis genes. But the function of the mating type locus in T. marneffei is not clear. The potential sex recombination might lead to problems in clinical, such as the evolution of increased resistance to antifungal drugs and virulence. To determine the mating type in a sample of 107 T. marneffei isolates and to explore the possible relationship between fungus virulence and mating type or source. We used PCR analysis to determine the distribution of mating type genes and also analyzed the relationship between mating type and isolated sources (including HIV-positive patients, HIV-negative patients, bamboo rats, and the environment). Further, the Drosophila melanogaster model of infection was used to compare the differences of virulence in mating type and sources. Our results showed the entire sample population of T. marneffei with an overabundance of MAT1-2 alleles, but with a higher ratio of MAT1-1 in the isolates from HIV-negative patients. However, no significant differences in the survival of the D. melanogaster infected neither with MAT1-1 (6.5 days) nor MAT1-2 (4 days) isolates. Similar results were also observed in virulence analysis tested with different sources of isolates. So, we found that all isolates bore single mating type idiomorphs and unequal distribution. The distribution of the MAT genes seems related to different sources. And the virulence differences are independent of mating type genotype and source.

Our work shows the entire sample population of 107 Talaromyces marneffei isolates with an overabundance of MAT1-2 alleles, but with a higher ratio of MAT1-1 in the isolates from HIV-negative patients. And fungus virulence is independent of mating type genotype and source in the Drosophila melanogaster model.

Combination therapy with itraconazole and terbinafine for phaeohyphomycosis caused by Exophiala spinifera: A case report and literature review.

Exophiala spinifera is a rare dematiaceous fungus causing cutaneous, subcutaneous and disseminated phaeohyphomycosis (PHM). Standard antifungal therapy for PHM is still uncertain. Here, we report a case of a Chinese male with PHM caused by E. spinifera, who received significant clinical improvement after the treatment with oral itraconazole and terbinafine. With the aim of evaluating the antifungal therapy for PHM caused by E. spinifera, a detailed review was performed.

Epidemiology and Clinical Findings of Tinea Capitis: A 23-Year Retrospective, Single-Center Study in Guangzhou, China.

BACKGROUND: Tinea capitis (TC) is one of the most common public health concerns due to its high incidence in preadolescent children. The epidemiological and clinical characteristics of TC vary depending on geographical regions and have changed over the past decades. OBJECTIVES: This study aimed to identify epidemiological changes in recent decades, including the prevalence and clinical and mycological characteristics of TC in southern China. METHODS: We conducted a retrospective study at the Department of Dermatology of Sun Yat-sen Memorial Hospital, Sun Yat-sen University from June 1997 to August 2020. RESULTS: We retrospectively evaluated 401 TC patients. Of these, 157 patients (39.2%) were preschool children aged 3-7 years and the majority were males. However, the prevalence in children under 3 years old is on the rise (from 19.67% during 1997-2010 to 32.49% during 2011-2020). Grey patches were the most common clinical pattern and mostly occurred in children (71.3%), while the proportion of grey patches and black dots was almost the same in adults. Although Microsporum canis (76%) was the most common causative organism, the number of the T. mentagrophytes complex, as a zoophilic fungus, has increased more than that of the anthropophilic fungi T. violaceum in the recent decade. There was a significant difference in the portion of sex among different age groups, and the gender difference was more notable in the adult group, which showed that the TC prevalence in females was 9 times that in males. In males, M. canis and the T. mentagrophytes complex were the two most common causative fungi, while M. canis and T. violaceum were the two most common causative fungi in females. Additionally, approximately 61.7% of black dot TCs occurred in females. For treatment, oral antifungal therapeutics were widely used in most patients with different treatment durations, although without a significant difference in efficacy (P = 0.106). CONCLUSIONS: In the last decade, the prevalence of TC in children under 3 years old increased, and boys dramatically outnumbered girls. In adults, the TC prevalence in females is nine times that in males, and most TCs occurring in females are presented as black dots. Moreover, the zoophilic T. mentagrophytes complex has replaced T. violaceum and is now the second most prevalent organism, followed by M. canis of TC.

Deep learning-based diagnosis models for onychomycosis in dermoscopy.

BACKGROUND: Onychomycosis is a common disease. Emerging noninvasive, real-time techniques such as dermoscopy and deep convolutional neural networks have been proposed for the diagnosis of onychomycosis. However, deep learning application in dermoscopic images has not been reported. OBJECTIVES: To explore the establishment of deep learning-based diagnostic models for onychomycosis in dermoscopy to improve the diagnostic efficiency and accuracy. METHODS: We evaluated the dermoscopic patterns of onychomycosis diagnosed at Sun Yat-sen Memorial Hospital, Guangzhou, China, from May 2019 to February 2021 and included nail psoriasis and traumatic onychodystrophy as control groups. Based on the dermoscopic images and the characteristic dermoscopic patterns of onychomycosis, we gain the faster region-based convolutional neural networks to distinguish between nail disorder and normal nail, onychomycosis and non-mycological nail disorder (nail psoriasis and traumatic onychodystrophy). The diagnostic performance is compared between deep learning-based diagnosis models and dermatologists. RESULTS: All of 1,155 dermoscopic images were collected, including onychomycosis (603 images), nail psoriasis (221 images), traumatic onychodystrophy (104 images) and normal cases (227 images). Statistical analyses revealed subungual keratosis, distal irregular termination, longitudinal striae, jagged edge, and marble-like turbid area, and cone-shaped keratosis were of high specificity (>82%) for onychomycosis diagnosis. The deep learning-based diagnosis models (ensemble model) showed test accuracy /specificity/ sensitivity /Youden index of (95.7%/98.8%/82.1%/0.809) and (87.5%/93.0%/78.5%/0.715) for nail disorder and onychomycosis. The diagnostic performance for onychomycosis using ensemble model was superior to 54 dermatologists. CONCLUSIONS: Our study demonstrated that onychomycosis had distinctive dermoscopic patterns, compared with nail psoriasis and traumatic onychodystrophy. The deep learning-based diagnosis models showed a diagnostic accuracy of onychomycosis, superior to dermatologists.

Antifungal Susceptibility Profiles of Olorofim (Formerly F901318) and Currently Available Systemic Antifungals against Mold and Yeast Phases of Talaromyces marneffei.

In vitro antifungal susceptibility profiling of 32 clinical and environmental Talaromyces marneffei isolates recovered from southern China was performed against olorofim and 7 other systemic antifungals, including amphotericin B, 5-flucytosine, posaconazole, voriconazole, caspofungin, and terbinafine, using CLSI methodology. In comparison, olorofim was the most active antifungal agent against both mold and yeast phases of all tested Talaromyces marneffei isolates, exhibiting an MIC range, MIC50, and MIC90 of 0.0005 to 0.002 µg/ml, 0.0005 µg/ml, and 0.0005 µg/ml, respectively.

Talaromyces marneffei infection in non-HIV-infected patients in mainland China.

BACKGROUND: Talaromyces marneffei, formerly known as Penicillium marneffei, is a significant emerging pathogenic fungus in Southeast Asia which can generate life-threatening systemic infections. Human immunodeficiency virus (HIV) infection is considered as the most underlying disease among systemic infections. However, infections due to T. marneffei without HIV are increasing in recent years. OBJECTIVES: Research the characteristics of T. marneffei infection in non-HIV individuals in mainland China. METHODS: In this study, we searched Pubmed, China National Knowledge Infrastructure (CNKI) and WanFang from inception to 31 December 2019 for studies reporting T. marneffei infection. Our research concentrates on non-HIV-infected cases and their epidemiology, clinical manifestations, laboratory findings, treatment methods and prognosis. RESULTS: T. marneffei infections in non-HIV individuals are increasing. Due to frequent present with atypical symptoms, these non-HIV-infected cases were usually misdiagnosed as other diseases, containing tuberculosis (80.7%), bacterial pneumonia (20.5%), lung cancer (5.1%) or other diseases (5.1%). CONCLUSIONS: T. marneffei infection in non-HIV individuals should be taken seriously. Their symptoms and signs are not typical. Accurate diagnosis and timely antifungal agent treatment is the key to the treatment for the disease.

Comparison of a fractional 2940-nm Er:YAG laser and 5% amorolfine lacquer combination therapy versus a 5% amorolfine lacquer monotherapy for the treatment of onychomycosis: a randomized controlled trial.

Onychomycosis is a fungal infection of the nail. The aim of this randomized controlled clinical trial was to compare the efficacy of 2940-nm Er:YAG laser treatment combined with a 5% amorolfine lacquer versus amorolfine monotherapy for treating onychomycosis. In this study, patients with onychomycosis of the great toenail were randomly assigned to a combination therapy group and a monotherapy group. In the combination therapy group, the included toenails were treated with a fractional 2940-nm Er:YAG laser at weeks 1, 2, 3, 4, 8, and 12, combined with a 5% amorolfine lacquer twice a week for 12 weeks, while in the monotherapy group, the included toenails were treated with only a 5% amorolfine lacquer twice a week for 12 weeks. The onychomycosis severity index (OSI) score and the mycological clearance rate (MCR) of the included toenails were assessed at baseline, week 12, and week 24. At weeks 12 and 24, the great toenails with mild and moderate onychomycosis in the combination therapy group showed obvious improvement and a greater decrease in OSI than those in the monotherapy group. At week 24, the toenails with mild and moderate onychomycosis in the combination therapy group also showed a better MCR. For the toenails with severe onychomycosis, little improvement was observed in either group at week 12 or week 24. In conclusion, fractional 2940-nm Er:YAG laser treatment combined with a 5% amorolfine lacquer is more effective than amorolfine monotherapy in short-term improvement of onychomycosis.

Activation of autophagy and IL-10 production are regulated by Jun N-terminal kinase 1 and 2 and p38 mitogen activated protein kinase signaling pathways during Talaromyces marneffei infection within dendritic cells.

Previous study have shown that Talaromyces marneffei (T. marneffei) induced activation of autophagy. Therefore, we explore signaling pathway that regulates activation of autophagy by intracellular signaling mechanisms during T. marneffei infection. Further, we examine c-Jun N-terminal kinase 1 and 2 (JNK1/2) and p38 signaling pathways that regulate IL-1ß and IL-10 production and activation of autophagy during T. marneffei infection in human dendritic cells (DCs). We found that T. marneffei induced activation of JNK1/2 and p38 in human DCs. Furthermore, the inhibition of JNK1/2 and p38 increased activation of autophagy and decreased the replication of T. marneffei in T. marneffei-infected human DCs. Moreover, IL-1ß secretion in T. marneffei-infected human DCs was dependent on JNK1/2 and autophagy pathways, whereas IL-10 secretion was dependent on JNK1/2, p38 and autophagy pathways. These data suggest that JNK1/2 and p38 pathways play critical roles in activation of autophagy, the multiplication of T. marneffei and subsequent cytokine production during T. marneffei infection.

Methylcitrate cycle gene MCD is essential for the virulence of Talaromyces marneffei.

Talaromyces marneffei (T. marneffei), which used to be known as Penicillium marneffei, is the causative agent of the fatal systemic mycosis known as talaromycosis. For the purpose of understanding the role of methylcitrate cycle in the virulence of T. marneffei, we generated MCD deletion (ΔMCD) and complementation (ΔMCD+) mutants of T. marneffei. Growth in different carbon sources showed that ΔMCD cannot grow on propionate media and grew slowly on the valerate, valine, methionine, isoleucine, cholesterol, and YNB (carbon free) media. The macrophage killing assay showed that ΔMCD was attenuated in macrophages of mice in vitro, especially at the presence of propionate. Finally, virulence studies in a murine infection experiment revealed attenuated virulence of the ΔMCD, which indicates MCD is essential for T. marneffei virulence in the host. This experiment laid the foundation for the further study of the specific mechanisms underlying the methylcitrate cycle of T. marneffei and may provide suitable targets for new antifungals.

Effect of Fonsecaea monophora on the Polarization of THP-1 Cells to Macrophages.

BACKGROUND: Chromoblastomycosis is a chronic, progressive fungal disease of the skin and subcutaneous tissue caused by a unique group of dematiaceous fungi. Fonsecaea monophora, a new species distinct from Fonsecaea pedrosoi strains, is the main pathogen responsible for chromoblastomycosis in south China. Macrophages can be polarized into two categories: classically activated and alternatively activated. OBJECTIVES: Little is known about the relationship between F. monophora and macrophage polarization. This study aimed to study the effect of F. monophora on the polarization of THP-1 cells to macrophages. METHODS: We established coculture systems of F. monophora and THP-1-derived macrophages in different activation states. RESULTS: F. monophora enhanced the phagocytosis by macrophages in the initially activated state and weakened the phagocytosis by classically activated macrophages without affecting that by alternatively activated macrophages. Classically activated macrophages had the strongest killing effect on F. monophora, while the initially activated macrophages had the weakest. The pathogen could not be rapidly cleared by any type of macrophage. F. monophora promoted the expression of proinflammatory cytokines and inhibited that of anti-inflammatory cytokines. CONCLUSIONS: F. monophora promoted the polarization of THP-1 cells to classically activated macrophages and inhibited that of THP-1 cells to alternatively activated macrophages.

Interplay of interferon-gamma and macrophage polarization during Talaromyces marneffei infection.

Talaromyces marneffei is an increasingly destructive dimorphic fungal pathogen in clinical settings that can cause lethal Talaromycosis. The activation of macrophages is known to be important for host defenses against T. marneffei, and these macrophages are known to be activated in two ways (polarization), known as M1 and M2. We investigated the plasticity of these polarizations, in order to understand if cross-conversion of macrophages may be possible even after they have been programmed. We conducted in vitro experiments using a murine macrophage cell line to investigate the ability of T. marneffei to activate these polarizations. The pre-polarized (M0) macrophage subsets were challenged with LPS as a control, and the sets of M1 markers (iNOS and CD86) and M2 markers (Arg-1 and CD206) were assessed for a possible cross-conversion among M1, M2 and M0 (unstimulated) populations. We found that either conidia or yeast forms of T. marneffei initiate the repression of Arg-1 in M2 cells with no change in the M1 subtype marker molecule iNOS. However, an additional IFN-γ stimulus caused the three macrophage groups to fully exhibit an LPS-induced M2 suppression and a shift to M1 from M0 and M2. We conclude that the conversion of macrophages is required for maintenance of sufficient iNOS production against this organism in the host. The cytokine environment is the key factor that manipulates the plasticity changes among macrophage subtypes. Furthermore, IFN-γ is a crucial host defense factor against pathogenic T. marneffei that has significant therapeutic potential to promote an M1 polarization phenotype.

Increased virulence of albino mutant of Fonsecaea monophora in Galleria mellonella.

Fonsecaea monophora has been the predominant pathogen of chromoblastomycosis in Southern China, but its pathogenic mechanism remains unclear. New models are needed to study this infection. In the current study, we examined the role of melanin on the pathogenicity of F. monophora in Galleria mellonella model using melanin and albino strain. Interestingly, the albino mutant strain displayed higher pathogenicity compared to the melanin stain and restoration of melanin of albino mutant could reverse the pathogenicity. Histopathology showed that inflammatory nodules were bigger than that infected with albino cells, which suggested that melanized cells could trigger a robust cellular immune response of G. mellonella than albino cells. The activated immune response in G. mellonella induced by melanized cells might explain the decreased virulence of melanized cells in larvae model. While further study was needed to gain full insights into the molecular immunological mechanism in G. mellonella activated by melanin.

Transcriptional profiling of macrophages infected with Fonsecaea monophora.

Fonsecaea monophora is a member of dematiaceous fungi capable of causing chromoblastomycosis through traumatic injury. However, little is known about the pathogenesis and early interactions between F. monophora and host. The aim of this study was to explore the potential mechanism of macrophages against F. monophora, especially the role of melanin during the pathogenic process. We carried out RNA sequencing based on the Illumina system. It showed that according to melanin contents, different strains of F. monophora induced different transcriptional profilings in macrophages. Functional analyses suggested the biological functions of differentially expressed genes were closely related to immune response, and the melanin might affect the interactions by regulating the MAPK signalling pathway of macrophages. Our results provide insights into the pathogenesis of infection by F. monophora conidia.

Talaromycosis (Penicilliosis) Due to Talaromyces (Penicillium) marneffei: Insights into the Clinical Trends of a Major Fungal Disease 60 Years After the Discovery of the Pathogen.

Talaromycosis (penicilliosis) is a major fungal disease endemic across a narrow band of tropical countries of South and Southeast Asia. The etiologic agent is a thermally dimorphic fungus Talaromyces (Penicillium) marneffei, which was first isolated from a bamboo rat in Vietnam in 1956, but no formal description was published. In 1959, Professor Gabriel Segretain formally described it as a novel species Talaromyces (Penicillium) marneffei, and the human pathogenic potential of the fungus in Mycopathologia. The first natural human case of talaromycosis (penicillosis) was reported in 1973 and involved an American minister with Hodgkin's disease who lived in Southeast Asia. Sixty years after the discovery of the pathogen, talaromycosis caused by T. marneffei is recognized as an important human disease with the potential to cause high mortality in the absence of proper diagnosis and prompt treatment. Talaromycosis remains a significant infectious complication in HIV/AIDS patients and in patients with other immune defects. The disease is being recognized with an increasing frequency well beyond the traditional endemic areas. The natural reservoirs of T. marneffei in wild rodents are well-defined, which links the ecology with the epidemiology of talaromycosis in endemic areas. There is an urgent unmet need for rapid and affordable point-of-care diagnostic tests. We also need more clinical studies to define the best therapeutic options for the management of talaromycosis patients.

Chromoblastomycosis.

Chromoblastomycosis (CBM), also known as chromomycosis, is one of the most prevalent implantation fungal infections, being the most common of the gamut of mycoses caused by melanized or brown-pigmented fungi. CBM is mainly a tropical or subtropical disease that may affect individuals with certain risk factors around the world. The following characteristics are associated with this disease: (i) traumatic inoculation by implantation from an environmental source, leading to an initial cutaneous lesion at the inoculation site; (ii) chronic and progressive cutaneous and subcutaneous tissular involvement associated with fibrotic and granulomatous reactions associated with microabscesses and often with tissue proliferation; (iii) a nonprotective T helper type 2 (Th2) immune response with ineffective humoral involvement; and (iv) the presence of muriform (sclerotic) cells embedded in the affected tissue. CBM lesions are clinically polymorphic and are commonly misdiagnosed as various other infectious and noninfectious diseases. In its more severe clinical forms, CBM may cause an incapacity for labor due to fibrotic sequelae and also due to a series of clinical complications, and if not recognized at an early stage, this disease can be refractory to antifungal therapy.

Role of autophagy in regulating the immune response of dendritic cells to Talaromyces marneffei infection.

Autophagy can regulate antimicrobial immunity. However, it is unknown whether autophagy mediates the immune response of dendritic cells (DCs) to Talaromyces marneffei (T. marneffei) infection. Therefore, to explore the relationship between autophagy and multiplication of T. marneffei and investigate whether ERK1/2 signaling pathway regulates activation of autophagy and TNF-α and IFN-γ secretion by intracellular signaling mechanisms during T. marneffei infection in human DCs. DCs were infected with T. marneffei for different times. First, we found that T. marneffei induced activation of autophagy and ERK1/2 in human DCs. Second, the inhibition of ERK1/2 suppressed activation of autophagy in T. marneffei-infected human DCs. Third, the suppression of ERK1/2 and autophagy decreased TNF-α and IFN-γ production and increased the proliferation of T. marneffei. These data suggest that ERK pathway plays vital regulatory roles in activation of autophagy and subsequent cytokine production during T. marneffei infection. Our data further indicate that autophagy is important in the regulation of the DC immune response to T. marneffei infection, thereby extending our understanding of host immune responses to the fungus.