Antifungal Resistance in Pulmonary Aspergillosis.

Aspergilli may cause various pulmonary diseases in humans, including allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), and acute invasive pulmonary aspergillosis (IPA). In addition, chronic colonization may occur in cystic fibrosis (CF). Aspergillus fumigatus represents the main pathogen, which may employ different morphotypes, for example, conidia, hyphal growth, and asexual sporulation, in the various Aspergillus diseases. These morphotypes determine the ease by which A. fumigatus can adapt to stress by antifungal drug exposure, usually resulting in one or more resistance mutations. Key factors that enable the emergence of resistance include genetic variation and selection. The ability to create genetic variation depends on the reproduction mode, including, sexual, parasexual, and asexual, and the population size. These reproduction cycles may take place in the host and/or in the environment, usually when specific conditions are present. Environmental resistance is commonly characterized by tandem repeat (TR)-mediated mutations, while in-host resistance selection results in single-resistance mutations. Reported cases from the literature indicate that environmental resistance mutations are almost exclusively present in patients with IA indicating that the risk for in-host resistance selection is very low. In aspergilloma, single-point mutations are the dominant resistance genotype, while in other chronic Aspergillus diseases, for example, ABPA, CPA, and CF, both TR-mediated and single-resistance mutations are reported. Insights into the pathogenesis of resistance selection in various Aspergillus diseases may help to improve diagnostic and therapeutic strategies.

Challenges towards management of CARD9-deficient patients with phaeohyphomycosis: A case report and case series study.

BACKGROUND: A number of recalcitrant phaeohyphomycosis cases with a life-threatening prognosis have been observed in CARD9-deficient patients, but little is known about the long-term management strategies that are effective for such intractable individuals. OBJECTIVES: To study the genetic and immunological mechanisms underlying recalcitrant phaeohyphomycosis and to share our clinical experiences regarding its treatment. PATIENTS/METHODS: Ten CARD9-deficient patients with recalcitrant phaeohyphomycosis admitted to our centre in the past two decades were followed-up, and their clinical presentations, laboratory findings, treatment and prognoses were analysed; one of them was a novel case of recalcitrant phaeohyphomycosis harbouring CARD9 mutations. Innate and adaptive immunological responses of patient-derived peripheral blood mononuclear cells were evaluated using ELISA and flow cytometry. RESULTS: We identified a total of seven CARD9 mutations in the ten analysed patients. Moreover, patient-derived cells exhibited a significant impairment of innate and adaptive immune responses upon fungus-specific stimulation. All the patients experienced recurrence and exacerbation; four of them died, two exhibited continued disease progress with unsatisfactory therapeutic efficacy, three showed obvious improvement under maintenance therapy, and only one achieved a clinical cure. CONCLUSIONS: Our study highlighted that otherwise healthy patients diagnosed with early-onset, unexplained and recalcitrant phaeohyphomycosis should be analysed for CARD9 mutations and immune deficiency. Thereafter, the length and choice of management remain challengeable and must be adjusted based on the clinical presentations and responses of patients over their lifetimes. Although continued posaconazole treatment may be the promising first-line therapy at present, novel strategies are worth exploring.

Dysbiosis of nail microbiome in patients with psoriasis.

Shifts in skin microbiome are considered to be involved in the pathogenesis of psoriasis. However, data on the microbial dysbiosis of nail psoriasis are scarce. In this study, we aim to investigate and characterize the nail bacterial and fungal microbiome in patients with psoriasis. Nail samples were collected prospectively from 36 subjects with nail psoriasis, 24 psoriatic subjects without nail involvement and 32 healthy controls. Amplicon sequencing was performed to evaluate the bacterial and fungal community compositions. Significant alterations in the bacterial microbiome were found in the nail samples of psoriatic patients. The unaffected nails in psoriatic patients were associated with higher bacterial diversity, and a higher relative abundance of Enhydrobacter, whereas nail psoriasis was correlated with a decreased relative abundance of Anaerococcus. Shifts in fungal community composition were reflected by a higher proportion of Malassezia in the unaffected nails of psoriatic patients and an increased proportion of Candida in psoriatic nails. Shifts in the nail microbiome in psoriasis suggest a potential role of microbes in the development of nail psoriasis. Future researches focusing on these microorganisms may help to explain the pathogenesis of psoriasis.

Ketoconazole 2% cream alters the skin fungal microbiome in seborrhoeic dermatitis: a cohort study.

BACKGROUND: Seborrhoeic dermatitis (SD) is a common chronic inflammatory dermatosis. Current theories on the pathogenesis of SD highlight the role of microbes on the skin surface. Ketoconazole is commonly used for the treatment of SD; however, there are limited data focusing on the effects of ketoconazole in shaping the skin microbiome in patients with SD. AIM: In this prospective cohort study, we used a high-throughput DNA sequencing method to characterize the cutaneous microbial communities of patients with SD before and after topical ketoconazole treatment. METHODS: In total, 30 patients with facial SD and 15 age- and sex-matched healthy controls (HCs) were enrolled in this study. Skin swabs were collected from SD lesional sites of the cheek at baseline, after ketoconazole treatment and 2 weeks post-treatment. DNA was extracted from skin samples. The bacterial 16S V3V4 rRNA and fungal internal transcribed spacer 1-5F regions were sequenced, and the microbial community compositions were analysed. RESULTS: Significantly lower bacterial and fungal diversities were detected at the lesional sites of facial SD compared with HCs. A decreased relative abundance of Cutibacterium and increased abundances of Malassezia and Staphylococcus were found in facial SD. Disease diversity was positively correlated with the relative abundances of Malassezia, Staphylococcus and Corynebacterium, while transepidermal water loss was negatively associated with the relative abundance of Cutibacterium. After ketoconazole treatment, fungal Shannon diversity and the relative abundances of Candida and Aspergillus were significantly increased at the lesional sites, and the relative abundance of Malassezia showed a decreasing trend. These changing trends were maintained until 2 weeks post-treatment. CONCLUSION: Facial SD showed lower fungal diversity accompanied by increased relative abundances of Malassezia and Staphylococcus and decreased relative abundance of Cutibacterium. Ketoconazole treatment reduced Malassezia and increased fungal diversity to restore skin microbial communities.

Characterisation of the nail microbiome in psoriatic and nonpsoriatic patients with onychomycosis.

BACKGROUND: Onychomycosis (OM) is the most common infectious nail disease, and it occurs frequently in patients with psoriasis. Microbial community shifts have been suggested to play a role in psoriasis and fungal infection occurrence. OBJECTIVES: To investigate and compare nail microbial community compositions in psoriatic and nonpsoriatic patients with OM. METHODS: Toenail samples were collected from nonpsoriatic patients with OM, psoriatic patients with nail psoriasis (NP) and OM, patients with only NP and healthy controls. Bacterial and fungal community compositions were analysed by amplicon sequencing of the V3-V4 regions of the 16S rDNA gene and the ITS1 region, respectively. RESULTS: Psoriatic OM patients had higher bacterial and fungal alpha diversities. Taxonomic analysis revealed a significantly lower relative abundance of Trichophyton rubrum (32.88% vs 82.18%, p < .001) and an increased trend of the abundance of Candida in psoriatic patients with OM than in nonpsoriatic patients. Nonpsoriatic patients with OM had a higher abundance of Staphylococcus than healthy controls (59.66% vs 45.76%, p < .05). Trichophyton, Alternaria and Malassezia could accurately differentiate psoriatic and nonpsoriatic patients with OM, with an area under the curve (AUC) of 0.86. The severity of OM was positively correlated with the relative abundance of Trichophyton rubrum. Further, Trichophyton was positively correlated with Staphylococcus and negatively correlated with Corynebacterium, Anaerococcus, Malassezia and Alternaria. CONCLUSIONS: The nail microbiome in psoriatic patients with OM has distinct bacterial and fungal signatures, suggesting that different dysbiosis is associated with the pathogenesis of OM in psoriatic and nonpsoriatic patients.

Ten decadal advances in fungal biology leading towards human well-being.

Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.

Primary Cutaneous Aspergillosis in a Patient with CARD9 Deficiency and Aspergillus Susceptibility of Card9 Knockout Mice.

PURPOSE: We describe a case of primary cutaneous aspergillosis caused by Aspergillus fumigatus, and elucidate the underlying genetic and immunological mechanisms. MATERIALS AND METHODS: Routine clinical and laboratory investigations were performed. Whole-exome sequencing of the patient's DNA suggested the presence of a CARD9 mutation, which was confirmed by Sanger sequencing. Innate and adaptive immunological responses of patient-derived CARD9-deficient cells were evaluated with ELISA and flow cytometry. Cutaneous and pulmonary aspergillosis models were established in Card9 knockout (KO) mice, which were compared with wild-type and immunosuppressed mice, to explore the pathogenesis and Aspergillus susceptibility. RESULTS: A 45-year-old man presented with a 37-year history of skin lesions on his face. A diagnosis of primary cutaneous aspergillosis was made through histopathology, immunohistochemistry, and tissue culture. Sanger sequencing of CARD9 showed a homozygous frame-shift mutation (c.819_820insG, p.D274fsX60), which led to the lack of CARD9 expression. Peripheral blood mononuclear cells from the patient showed selective impairment of proinflammatory cytokines, and Th1-, Th17-, and Th22-associated responses upon fungus-specific stimulation. The cutaneous aspergillosis model established in Card9 KO mice presented with persistent infection, with fungal germs and short hyphae in tissue, consistent with the patient's lesions. Skin lesions in immunosuppressed mice were more severe, and led to death. Unlike our patient, Card9 KO mice were relatively susceptible to pulmonary aspergillosis, with reasons to be investigated. CONCLUSIONS: This is, to our knowledge, the first report that links cutaneous aspergillosis to CARD9 mutation. This work enriches both the phenotypic spectrum of CARD9 deficiencies and the genetic background of cutaneous aspergillosis.

The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin-fixed paraffin-embedded tissues.

OBJECTIVE: To develop a comprehensive diagnostic system for mucormycosis from formalin-fixed paraffin-embedded tissues, consisting of own-designed real-time polymerase chain reaction (PCR) assays, fluorescence in situ hybridisation, and immunohistochemical staining. METHODS: We designed 11 primers and probes for specific real-time PCR assays based on genome sequences, and validated the specificity by Aspergillus, Fusarium, Scedosporium, Lomentospora, Cryptococcus and Candida species. Formalin-fixed paraffin-embedded (FFPE) tissues from forty-four mouse model infected by above fungi were collected and extracted DNA by laser capture microdissection (LCM) and direct extraction methods for real-time PCR assays. In addition, seventeen clinical specimens histopathologically proven for mucormycosis were included for specific detection with the new diagnostic system. RESULTS: The real-time PCR assays allowed detection of a minimum of 10 CFU/ml equivalent gDNA of each species. No cross-reaction with gDNA among species was noted. From mouse model specimens, the sensitivity of real-time PCR in samples extracted with LCM versus direct extraction method was 100% versus 91.43% at Mucorales level and 80% versus 45.71% at species level, respectively. The specificity was 100%. From clinical samples, LCM combined with real-time PCR can test 88.24% (15/17) of Mucorales. Sensitivities of fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were 70.59% and 41.18%, respectively. Combined LCM-RT-PCR, FISH and IHC yielded positive results in all samples. CONCLUSIONS: The combination diagnostic system we developed is a culture-independent and robust method which enables rapid species identification from FFPE tissues for timely diagnosis of mucormycosis.

Molecular genotyping of Candida albicans isolated from different sites may trace the etiological infection routes: Observation in China.

BACKGROUND: Invasive candidiasis is a growing concern worldwide, especially in immunocompromised patients, including ICU patients. OBJECTIVES: As Candida albicans is the leading cause of candidaemia, it is important to investigate the evolution of C. albicans in patients with candidaemia. METHODS: We analysed 238 strains of C. albicans isolated from different body sites. Antifungal susceptibility testing, CAI loci genotyping and multilocus sequence typing (MLST) of all isolates were performed. The relationships among the total isolates that differed in sequence at only one of the seven housekeeping gene loci were analysed using eBURST. RESULTS: Multilocus sequence typing analysis in 238 isolates by combining seven housekeeping alleles revealed 175 diploid sequence types, in which 84 were newly identified. eBURST analysis for these data recognised 19 clonal complexes (CCs) and 79 singletons. Besides, seventy-three CAI genotypes were identified. Blood isolates showed maximum genotypes (49), and the dominant genotypes were CAI 17-21 and CAI 21-21. Oral isolates possessed 25 CAI genotypes, and the dominant genotypes were CAI 17-21 and CAI 21-21 as well. Since isolates with CAI allele numbers <30 showed easier transmission, CAI 17-21 and CAI 21-21 were the most frequently transmitted. Finally, the CAI genotypes were classified into six groups. CONCLUSIONS: This work revealed the oral and blood strains isolated from the patients with candidaemia in ICU shared the identical dominant CAI genotypes. Our data expanded the C. albicans MLST database and helped with understanding the evolution and spread of invasive candidiasis.

Comparative genomics of opportunistic Phialophora species involved in divergent disease types.

BACKGROUND: Black opportunists Phialophora verrucosa complex species can cause different disease types in competent and in immunocompromised individuals, but are remarkably overrepresented in CARD9-related infections. OBJECTIVES: To better understand the ecology and potential pathogenicity of opportunistic Phialophora species and reveal eventual genetic parameters associated with the behaviour in vivo and genetic profiles in patients with CARD9 immunodeficiency. METHODS: Genomes of 26 strains belonging to six species of the Phialophora verrucosa complex were sequenced. Using multilocus analysis, all environmental and clinical strains were identified correctly. We compared the genomes of agents from different disease types among each other including CARD9 immunodeficiency. RESULTS: We obtained genome sizes of the 26 Phialophora strains ranged between 32 and 37 MB. Some species showed considerable intraspecific genomic variation. P americana showed the highest degree of variability. P verrucosa was variable in CAZy enzymes, whereas P americana varied in PKS-related genes. Phialophora species, particularly P verrucosa, are relatively frequent in patients with CARD9-related immunodeficiency. Different mutations in the CARD9 gene seem to increase susceptibility for infection by different groups of species, that is either Candida, dermatophytes or black fungi. A number of patients with chromoblastomycosis revealed an as yet unknown CARD9 mutation. TNFα impairment was prevalent in patients with CARD9 infections, while CBM patients were invariably IFNγ. CONCLUSIONS: From genomic investigations, the known virulence factors between clinical and environmental strains did not reveal any significant difference. Phialophora complex has an equal chance to cause infection in humans, either healthy or CARD9-impaired.

Molecular and MALDI-ToF MS differentiation and antifungal susceptibility of prevalent clinical Fusarium species in China.

BACKGROUND: Fusarium species are emerging causative agents of superficial and disseminated human infections. Early diagnosis and treatment contribute to better prognosis of severe infection. OBJECTIVES: To detect the effectiveness of matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) for Fusarium identification, and evaluate the susceptibility profiles to clinical available antifungals. METHODS: All 203 clinical Fusarium isolates and 25 environmental isolates were identified by using translation elongation factor 1-alpha (TEF1) and RNA polymerase subunit II (RPB2) sequencing and MALDI-ToF MS. Antifungal susceptibility testing was determined by a microdilution method following the CLSI approved standard M38-A3 document. RESULTS: Correct identification rates at the species and genus levels were 89.04% (203/228) and 95.18% (217/228), respectively, using Bruker Filamentous Fungi Library 1.0 combined with the novel database. Seven species complexes with 19 Fusarium species were identified, including F. solani (59.21%, n = 135), F. verticillioides (17.54%, n = 40), F. proliferatum (6.58%, n = 15) and F. oxysporum (4.39%, n = 10). Four uncommon species complexes (F. incarnatum-equiseti SC, F. dimerum SC, F. redolens SC and F. sporotrichioides SC) were also identified. A high degree of antifungal resistance was observed. Fusarium isolates exhibited lower MICs to luliconazole and terbinafine compared with amphotericin B and voriconazole, which in turn were significantly more active than amorolfine, fluconazole and itraconazole. CONCLUSIONS: MALDI-ToF MS showed good performance in Fusarium species with an adapted Bruker library and expanded database. Fusarium isolates exhibited lower MICs to luliconazole and terbinafine compared to amphotericin B and voriconazole.

Tinea Capitis by Microsporum canis in an Elderly Female with Extensive Dermatophyte Infection.

Tinea capitis is a type of dermatophyte infection primarily affecting children. We report a case of an elderly woman with well-controlled diabetes mellitus presenting with a six-month history of erythema with yellow crusts on her scalp and extensive erythematous patches with scales on the body skin. She adopted a stray cat before the disease onset. Dermoscopic findings and manifestation under the Wood's lamp favoured the diagnosis of tinea capitis. Further microscopic examinations of her scalp, including direct KOH and fluorescence stain examination, fungal culture and polymerase chain reaction sequencing identification confirmed the diagnosis of tinea capitis caused by Microsporum canis. Treatment with oral terbinafine was effective. Adult tinea capitis is often misdiagnosed due to its rarity and atypical presentation. However, in some regions, the incidence of tinea capitis in immunocompetent adults is rising which requires the awareness of clinicians. A thorough history (including the animal contacting history), physical examination and further mycological examinations are required for diagnosis. Trichophyton violaceum is the most common dermatophyte species in most regions while adult tinea capitis caused by Microsporum canis is less common. Terbinafine, griseofulvin and itroconazole have been reported to be effective drugs for the treatment of tinea capitis, and terbinafine can be considered as systemic treatment in elderly patients with comorbidities to reduce the drug-drug interaction.

Disseminated Cryptococcosis Presenting as Cellulitis Diagnosed by Laser Capture Microdissection: A Case Report and Literature Review.

Disseminated cryptococcosis primarily affects immunosuppressed patients and has a poor outcome if diagnosis and treatment are delayed. Skin lesions are rarely manifest causing misdiagnosis. We present a case of cryptococcal cellulitis with severe pain in a kidney transplant recipient on long-term immunosuppressive therapy. Multiple organs were involved, and there was cutaneous dissemination of the lesions. Histopathology revealed abundant yeast-like cells with wide capsular halos in subcutaneous tissue, suggesting Cryptococcus spp. infection. Laser capture microdissection (LCM)-PCR on skin biopsies confirmed Cryptococcus neoformans var. grubii. A literature review of 17 cases of disseminated cryptococcosis with cutaneous cellulitis or panniculitis in HIV-negative individuals found that over half the patients (52.9%, 9/17) had a history of glucocorticoid therapy, and that the most common site was the legs (76.5%, 13/17). C. neoformans was the main pathogenic species, accounting for 88.2% (15/17) of cases. Fungal cellulitis should be included in the differential diagnosis of cellulitis that fails to respond to antimicrobial therapy in HIV-negative immunosuppressed individuals. Non-culture-based molecular techniques aid in rapid pathogen identification in histologically positive, unculturable specimens.

Diagnostic value of fluorescein-labeled chitinase staining in formalin-fixed and paraffin-embedded tissues of fungal disease.

Common histopathologic techniques are used to diagnose fungal infections, but the diagnostic identification of mycoses in tissue specimens is often difficult, particularly when fungi rarely occur in a specimen. The aim of this study was to evaluate the application of fluorescein-labeled chitinase staining to formalin-fixed and paraffin-embedded (FFPE) tissues. We studied 79 archival FFPE tissues from patients diagnosed with fungal disease, including 38 cases of sporotrichosis and 41 cases of other fungal infections. The tissue sections were subjected to periodic acid-Schiff (PAS) staining, Gomori's methenamine silver (GMS) staining, and fluorescein-labeled chitinase staining to detect fungal elements. Culture- and/or hematoxylin-eosin-positive samples were used to estimate the diagnostic sensitivity of each staining method, with the results showing that PAS, GMS, and fluorescein-labeled chitinase staining had sensitivities of 50.6, 70.9, and 68.4%, respectively. The three staining results were the same for all fungal infections except for sporotrichosis and chromoblastomycosis. Fluorescein-labeled chitinase staining exhibited high sensitivity in cases of sporotrichosis and poor performance in detecting muriform cells of chromoblastomycosis. On the whole, the sensitivity of fluorescein-labeled chitinase staining was greater than that of PAS and similar to that of GMS staining. Therefore, the results of our study suggest that fluorescein-labeled chitinase staining is a potentially useful diagnostic tool in the diagnosis of fungal infections.

Phaeohyphomycosis caused by Phialophora americana with CARD9 mutation and 20-year literature review in China.

BACKGROUND: Phaeohyphomycosis is a chronic cutaneous, subcutaneous or systemic mycotic infection caused by various dematiaceous fungi. The diverse clinical manifestations and poor prognosis of phaeohyphomycosis necessitate studies on it to better recognise the disease and improve its management. OBJECTIVES: To investigate the epidemiology, aetiology, diagnosis, treatment and prognosis of phaeohyphomycosis in China over the past 20 years, and to study the first case of phaeohyphomycosis caused by Phialophora americana and the genetic and immunological mechanisms. PATIENTS/METHODS: Clinical and laboratory findings of the case were studied, and the patient's DNA was sequenced for CARD9, followed by immunological studies using patient's PBMCs. Cases of phaeohyphomycosis in China from 1998 to 2018 in both the Chinese and English literature were collected and analysed, including 45 articles and 46 patients. RESULTS: We confirmed the patient holding a homozygous frameshift mutation of CARD9, which led to impairment of pro-inflammatory cytokine production, and lower Th17- and Th22-associated responses upon fungus-specific stimulation. From the literature review, we revealed that the clinical presentations of phaeohyphomycosis were diverse. Diagnoses were established mainly on the basis of histopathology and fungal culture. Oral itraconazole, voriconazole, and posaconazole are the first choices for treatment, and a combination with surgical excision is also recommended. CONCLUSIONS: Our study establishes that obtaining detailed histories is vital for understanding the immune state and that patients with recurrent or chronic phaeohyphomycosis in the absence of known immunodeficiencies should be tested for CARD9 mutations. We hope our findings will aid clinicians in the diagnoses and treatment of such infections.

Disfiguring Mucor irregularis Infection Cured by Amphotericin B and Itraconazole: A Case Report and Treatment Experience.

We report a case of primary cutaneous mucormycosis caused by Mucor irregularis. A 52-year-old male farmer was presented to our hospital with a history of progressive nodule and plaque with ulceration on the face for two and a half years. Broad, aseptate hyphae were seen in direct KOH examination and biopsy. Fungal culture showed light yellow filamentous colonies. The rRNA sequencing revealed M. irregularis was the responsible fungus. Amphotericin B in gradually increasing dose and itraconazole were administered. When the cumulative dose of amphotericin B was 1500 mg, the skin lesion improved significantly with remaining scars on the face. Then, the patient received sequential oral itraconazole treatment for 8 months. There was no recurrence up to now through follow-ups.

Impairment of Immune Response against Dematiaceous Fungi in Card9 Knockout Mice.

Dematiaceous fungi are a large group of pathogens that can cause a wide range of diseases in both immunocompetent and immunocompromised hosts. Based on our previous finding of caspase recruitment domain-containing protein 9 (CARD9) mutations in patients with subcutaneous phaeohyphomycosis caused by Phialophora verrucosa (P. verrucosa), we further investigated the exact role of CARD9 in the pathogenesis of phaeohyphomycosis using Card9 knockout (Card9 KO) mice. We showed that Card9 KO mice are profoundly susceptible to P. verrucosa infection compared with wild-type mice, reflected by significantly more severe footpad swelling, higher fungal burden, lower survival, and systemic dissemination. The inability of Card9 KO mice to control P. verrucosa infection was associated with lack of Th17 differentiation and reduction of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-17A levels in footpad homogenates. In vitro experiments showed a defect of fungal conidia killing and pro-inflammatory cytokine production in Card9 KO bone marrow-derived macrophages and dendritic cells. Furthermore, ex vivo coculture and in vitro T cell differentiation assay demonstrated that Card9 signaling pathway acts indispensably on differentiation of Th17 cells. In conclusion, our findings suggest that CARD9 mediate the innate immune and Th17-mediated adaptive immune responses against dematiaceous fungal infections at the early stage of infection.