ABSTRACT The clinical management of invasive aspergillosis (IA) is limited by the scarcity of antifungal agents and the emergence and spread of drug resistance, which impairs therapeutic efficacy and poses a significant public health threat. Olorofim, a first-in-class clinical antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapeutic option for IA. It shows activity against several filamentous fungi, including Aspergillus spp., both in vitro and in vivo . Olorofim-resistant Aspergillus fumigatus isolates, characterized by various amino acid substitutions (with a hotspot at G119), have been generated under laboratory conditions through olorofim exposure. However, the potential olorofim resistance mechanisms in Aspergillus flavus , the second-most common pathogen causing IA globally, remain entirely unexplored. In this study, we revealed that the residue G118 in A. flavus , conserved in many filamentous fungi, is homologous to G119 in A. fumigatus by DHODH amino acid sequence alignment. Using a CRISPR-based point mutation approach, we further confirmed that substitutions at the DHODH G118 residue confer acquired olorofim resistance in A. flavus . Additionally, we established a Galleria mellonella infection model and observed that these resistant isolates exhibited no obvious fitness cost, and olorofim treatment was ineffective against infections caused by DHODH G118 mutants. Molecular docking models further indicated that substitutions at the G118 locus may introduce steric hindrance, reducing the binding affinity between olorofim and the DHODH active site and thereby driving resistance. IMPORTANCE Invasive aspergillosis, a life-threatening infection in immunocompromised patients, is increasingly difficult to treat due to limited antifungal options and rising resistance. Olorofim, a novel antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapy. While resistance in Aspergillus fumigatus has been linked to DHODH G119 mutations, the mechanisms in the globally prevalent pathogen Aspergillus flavus remained unknown. Using CRISPR-based mutagenesis, we demonstrate that amino acid substitutions at G118 confer olorofim resistance, supported by molecular docking showing reduced drug binding. Importantly, olorofim failed to protect larvae infected with resistant mutants in an infection model. These findings provide essential insights for proactively monitoring and managing olorofim resistance in Aspergillus flavus , aiding clinical and public health responses. This study is registered with ClinicalTrials.gov as NCT05101187 and NCT06969703 .

OBJECTIVES:To investigate the species distribution and antifungal susceptibility profiles of clinical mould isolates from China to olorofim, manogepix, amphotericin B, triazoles and echinocandins. METHODS:Isolates were collected from patients at 10 tertiary hospitals across China between 2019 and 2024. Species identification was performed by sequence analysis. Antifungal susceptibility testing was performed according to the CLSI reference methods. The cyp51A, cyp51B and hmg1 genes from triazole-resistant isolates were amplified to identify mutations associated with resistance. RESULTS:Aspergillus spp. (92.02%) remained the most prevalent pathogens, followed by Fusarium spp. (4.18%) and Mucorales (1.90%). The proportion of non-A. fumigatus isolates showed an increasing trend. The majority of Aspergillus spp. were susceptible/WT to triazoles (97.52%), with posaconazole showing the highest potency. Among the triazole-resistant Aspergillus isolates, two harboured cyp51A mutations (TR46/Y121F/T289A, G441S) and one carried an hmg1 mutation (V827L). Notably, the novel antifungals olorofim and manogepix were highly potent against most tested moulds, including triazole-resistant Aspergillus isolates. For Fusarium spp., manogepix showed low MECs, whereas olorofim and triazoles showed higher and more species-specific MICs. Both novel agents showed high MIC/MECs against Mucorales isolates, and triazole MIC distributions varied markedly between species. CONCLUSIONS:A. fumigatus sensu stricto remained the predominant pathogen while non-A. fumigatus moulds became increasingly prevalent. Triazole resistance among clinical Aspergillus isolates was uncommon but was associated with cross-resistance and target gene mutations. Novel antifungals olorofim and manogepix demonstrated potent in vitro activity against a broad range of clinical moulds, including triazole-resistant Aspergillus isolates.

The skin microbiome plays an important role in aging, yet most aging biomarkers predominantly focus on gut bacteria, overlooking the skin microbial communities, especially its fungal component. To comprehensively profile the skin bacterial and fungal microbiome across age, sex, and anatomical sites (sun-exposed forehead vs. non-sun-exposed back) and develop an integrated microbial model for age prediction. A total of 160 skin swabs from 80 healthy individuals stratified into four age groups (centered at 10, 30, 50, 70 years) were conducted by DNA sequencing for microbial analysis. An age-predictive model was built using a random forest classifier trained on bacterial and fungal composition data. We found clear age- and sex-specific differences in the skin microbiome. Fungal diversity was significantly higher in females, while bacterial diversity decreased markedly around age 30 in both sexes. Malassezia dominated fungal communities; its abundance peaked at 30 years, declining with age, especially on female foreheads. Age-dependent shifts occurred in dominant Malassezia species (e.g., M. globosa in children, M. arunalokei in the elderly). Bacterial communities shifted from diverse childhood profiles (e.g., Pseudomonas, Streptococcus) to Cutibacterium dominance in young adulthood, which declined in older individuals. Correlation analysis revealed stronger age-microbe associations in males. Finally, we developed a predictive model using four key microbial markers—Lactarius (fungus), Chryseobacterium, Gordonia, and Psychrobacter—that showed good performance in age-group classification (AUC = 0.97). Collectively, these findings reveal distinct age- and sex-related patterns in the skin microbiome, highlight the importance of including fungi in microbiome studies, and demonstrate the potential of microbial profiles as candidate age-associated signatures.

IntroductionChromoblastomycosis (CBM) is a chronic, neglected tropical fungal infection. Its immunopathogenesis, particularly the mechanism underlying its chronicity, remains poorly understood.MethodsWe performed single-cell RNA sequencing (scRNA-seq) on lesional skin from a CBM patient, followed by comprehensive bioinformatics analyses. We then used multiplex immunofluorescence (mIF) to validate CD4+ T cell exhaustion in CBM patient lesions and the mouse model of Fonsecaea pedrosoi infection.ResultsWe identified a significantly expanded population of exhausted CD4+ T cells within the patient’s lesions, which exhibited high co-expression of inhibitory receptors (PD-1, TIM-3, LAG-3) and functional impairment. Trajectory inference suggested a differentiation path from naive towards exhaustion within the chronic inflammatory environment. Cell-cell communication analysis implicated monocytes/macrophages (MoMacs) as key drivers of this process via persistent antigen presentation and ligand-receptor interactions such as CTLA4-CD80/86 and LGALS9-CD44. The accumulation of exhausted CD4+ T cells was confirmed in human CBM lesions by multiplex immunofluorescence (mIF), and the progressive development of exhaustion was recapitulated in the mouse model of Fonsecaea pedrosoi infection.DiscussionOur findings establish CD4+ T cell exhaustion as an important mechanism underlying the chronicity of chromoblastomycosis, revealing a new immunopathological perspective for this neglected disease.

Invasive candidiasis caused by Nakaseomyces glabratus is of great concern due to high morbidity and mortality, especially antifungal resistance. To identify genomic signatures, which significantly link to drug-resistance, is of great significance in combating this lethal disease. In this study, we performed whole genome analysis on 109 clinical strains of N. glabratus which had been isolated from multi-centres in China. By using genome-wide association studies (GWAS), genomic signatures, including several PDR1 mutations and genes encoding GLEYA-containing proteins, were identified to be significantly linked to drug-resistance. With the strategy of feature-selection combining machine-learning (ML), more relevant genomic signatures and potential resistance determinants were identified, including Y682C and I380L mutations in PDR1 which were further confirmed to confer triazole-resistance by gene editing technology. We believe that the ML-based feature selection (MLFS) strategy, which is based on a comprehensive understanding of genomic characteristics as described in this study, shows excellent capacity to predict resistance and potential resistance determinants in N. glabratus.

BACKGROUND:Tinea pedis is a type of dermatophytosis that affects the superficial layers of the skin on feet. Limited data are available on the skin microbiome composition in affected patients and its changes following topical antifungal therapy. OBJECTIVES:To evaluate the clinical and microbiological effects of topical ketoconazole 2% cream (KTZ) and miconazole nitrate 2% cream (MCZ) using standardised clinical scoring and amplicon sequencing. METHODS:A total of 42 patients with tinea pedis and 28 healthy controls were enrolled. Skin swabs were collected from lesional sites (interdigital or heel) at baseline, after 4 weeks of treatment, and 2 weeks post-treatment. DNA was extracted from the samples, and the bacterial 16S rRNA (V3-V4 region) and fungal ITS1-5F regions were sequenced to analyse microbial community composition. RESULTS:Both KTZ and MCZ led to comparable clinical improvement. However, the KTZ group showed faster symptom resolution and a higher sustained improvement rate during follow-up. Treatment with either antifungal effectively reduced the abundance of pathogenic Trichophyton species to levels similar to those in healthy controls, thereby contributing to partial recovery of the overall fungal community structure. In parallel, the bacterial profile became more dispersed, with notable shifts observed in bacterial genera such as Staphylococcus and Corynebacterium following treatment. CONCLUSION:Topical antifungal therapy with KTZ or MCZ effectively improved the symptoms of tinea pedis, diminished the pathogenic fungal load and altered both fungal and bacterial community compositions. However, only partial restoration of the mycobiome was achieved, and the bacterial profile, especially in the interdigital region, showed a lack of bacterial normalisation. These findings highlight the need for further studies to assess long-term outcomes and to explore microbiome-targeted strategies addressing both bacterial and fungal components.

Our study is the first to compare fungal microbiome changes in patients with atopic dermatitis (AD) before and after treatment with upadacitinib and dupilumab. We found that upadacitinib more effectively restores the fungal microbiome to levels observed in healthy individuals, which may contribute to its superior efficacy in head-and-neck dermatitis (a subtype of AD). The results enhance our understanding of how JAK inhibitors influence the skin microbiome in AD.

Vulvovaginal candidiasis (VVC) is an inflammation caused by Candida albicans with a higher recurrence rate in individuals deficient in Card9. This study aimed to elucidate the mechanisms underlying this increased susceptibility. Estrogen-treated Card9-/- mice infected with C. albicans were used to model Card9 deficiency-related VVC. Our findings indicate that Card9 deficiency leads to a reduction in Th17 cells, interleukin (IL)-17-producing γδ T cells, and IL-17A secretion, weakens epithelial tight junctions, and reduces antimicrobial peptide secretion, leading to persistent fungal invasion. This persistent invasion results in excessive neutrophil recruitment and activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) and absent in melanoma 2 inflammasomes (AIM2), causing mucosal damage. In conclusion, Card9 deficiency compromises the vaginal epithelial barrier, prolongs C. albicans infection, and increases inflammation, highlighting the critical role of Card9 in maintaining immune function of vaginal mucosa.

Fungi in the genus Fusarium are plant pathogens but are also capable of causing a wide range of diseases in humans. The intrinsic multi-drug resistance of Fusarium often leads to a poor clinical outcome in patients with severe immune disorders. Olorofim, a member of the orotomide class, is a novel type of antifungal drug that interferes with pyrimidine biosynthesis by inhibiting dihydroorotate dehydrogenase and thereby prevents growth and cell division. In this study, the in vitro activity of olorofim was evaluated against 253 Fusarium isolates, of which 228 isolates belonging to the prevalent complexes involved in human infection, F. solani species complex (SC) and F. fujikuroi SC. All Fusarium isolates underwent species-level identification via multi-locus sequence typing (MLST) targeting RPB1 , RPB2 , and TEF1 loci. Antifungal susceptibility testing was performed using a CLSI M38, 3rd ed. broth microdilution for olorofim. The geometric mean of the MICs of olorofim for all 253 isolates was 0.581 µg/mL, ranging from 0.015 µg/mL to >16 µg/mL. Olorofim demonstrated high MICs against F. solani SC, whereas greater potency (lower MICs) was observed against F. fujikuroi SC. Clinical isolates tended to have higher MIC values than environmental isolates, but this pattern was not consistent across all species complexes. Overall, olorofim demonstrated moderate in vitro activity against Fusarium isolates, suggesting it might be a potential candidate for treating fusarioses caused by multidrug-resistant strains.

Trichophyton species, the leading cause of dermatophytosis globally, are increasingly resistant to antifungal treatments, concerns about effective management strategies. In light of the absence of established resistance criteria for terbinafine and azoles, coupled with a dearth of research on resistance mechanisms in Trichophyton, antifungal susceptibility and drug resistance gene diversity were analyzed across 64 T. mentagrophytes, 65 T. interdigitale, and 2 T. indotineae isolates collected in China between 2001 and 2024 and 101 published T. indotineae strains. Analyses of the minimum inhibitory concentrations (MICs) of terbinafine, itraconazole, voriconazole, posaconazole, and isavuconazole revealed a concerning increase in T. indotineae with terbinafine resistance, including two novel isolates from China. Compared with T. interdigitale, T. mentagrophytes presented higher terbinafine MICs but similar azole susceptibility. Notably, 27 T. interdigitale isolates were classified as non-wild-type for terbinafine. Genetic diversity was analyzed for the SQLE, CYP51A and CYP51B gene. Specifically, T. indotineae isolates presented SQLE protein changes linked to terbinafine resistance. SQLE diversity was linked to terbinafine sensitivity, whereas alterations in CYP51A were associated with itraconazole sensitivity, with notable statistical significance evident across various protein isoforms. The relationship between protein diversity and drug sensitivity is presented in detail. Together, these findings highlight a growing prevalence of antibiotic resistance among Trichophyton and identify potential target genes for new therapies, underscoring the need for ongoing monitoring and offering directions for novel therapeutics.

OBJECTIVES:Tinea capitis remains a common fungal infection in children worldwide. Species identification is critical for determining the source of infection and reducing transmission. In conventional methods, macro- and microscopic analysis is time-consuming and results in slow fungal growth or low specificity. We propose a rapid real-time diagnostic PCR method that allows species-specific identification of dermatophytes, including the Microsporum canis complex, Trichophyton mentagrophytes complex, Trichophyton rubrum complex and Trichophyton tonsurans, in patients with tinea capitis. METHODS:Hair and scrapings samples were collected from 231 patients with tinea capitis who were positive for fungal elements via direct microscopy with potassium hydroxide. Each sample was subjected to a two-step real-time PCR (RT-PCR) assay, which was designed on the basis of differences in the DNA fragments of the internal transcribed spacer (ITS) and β-tubulin covering the Microsporum canis complex, T. mentagrophyte complex, T. rubrum complex, T. tonsurans, T. verrucosum, T. schoenleinii and N. gypseum. RESULTS:In total, 186/231 samples (80.52%) were positive for fungal culture. The two-step RT-PCR was positive in 215/231 samples (93.07%), among which 179 were culture positive. The combined efficacy was 96.81%, which was significantly different when the RT-PCR assays were performed in parallel with fungal culture. A total of 126 samples (54.55%) were identified as Microsporum canis by fungal culture, among which the positive rate of M. canis complex RT-PCR was 97.62% (123/126). A total of 45 samples were negative for fungal culture, of which 80.0% (36/45) were positive by RT-PCR, and the percentage of M. canis complex-positive samples was 53.33% (24/45). The RT-PCR assays were negative for 16/231 samples, among which 7 were culture positive, including M. canis (n = 3), T. violaceum (n = 3) and N. gypseum (n = 1). CONCLUSION:We developed a new diagnostic assay system using a rapid real-time TaqMan PCR assay with specific primers that can be applied in routine laboratory practice for hair and skin samples of tinea capitis to detect dermatophytes and increase diagnostic efficiency.

OBJECTIVE:Trichophyton mentagrophytes complex species mainly cause superficial infections, with a high global incidence and affecting the quality of life of patients. The taxonomic identification between closely related species within the T. mentagrophytes complex remains problematic, especially between T. mentagrophytes and T. interdigitale. This study aimed to elucidate the species boundaries within the T. mentagrophytes complex. METHODS:We performed a phylogenetic tree, principal component analysis and population structure analysis based on whole genome single-nucleotide polymorphism (SNP) data of 157 T. mentagrophytes complex strains. The mating types, phenotypic and physiological characteristics of different populations of strains were detected to delimit the species within the complex. RESULTS:The phylogenomic analysis showed that the 157 T. mentagrophytes complex strains were divided into five populations and clustered into three major clades, namely the T. tonsurans (population I), T. interdigitale (populations II, III and V) and T. mentagrophytes (population IV) clades. T. interdigitale population III was significantly different from T. interdigitale population II in terms of nucleotide diversity, mating types, types of clinical disease caused and keratinolytic ability but similar to those of T. mentagrophytes population IV. CONCLUSIONS:T. tonsurans, T. interdigitale and T. mentagrophytes could be regarded as independent species and all strains were divided into five populations. The finding that T. interdigitale population III strains (T. mentagrophytes genotype Tm-II*) are reclassified as T. interdigitale based on whole-genome analysis is notable and clarifies previous confusion in clinical microbiology labs.

BACKGROUND:Inherited genetic deficiencies in the Caspase-associated recruitment domain-containing protein 9 (CARD9) lead to increased susceptibility of patients to opportunistic melanized fungi. Such infections are recalcitrant, and the fungus possibly acquires resistance under therapy. OBJECTIVE:To evaluate differences of in vitro antifungal susceptibility of strains of melanized fungi originating from patients with CARD9 deficiency versus strains from chronic patients with unclear genetic background. METHODS:We analyzed a total of 118 isolates, including 33 from patients with CARD9 deficiency, 80 from chronic patients with other undefined immunological features, and 5 environmental strains, all collected between 1997 and 2021. All isolates were identified by sequencing the ITS spacer of the rDNA operon. Broth microdilution susceptibility tests were performed according to CLSI guidelines (M38-A3document). RESULTS:MIC ranges of strains from infected patients having CARD9 deficiency and other individuals were mostly similar. However, comparing these two groups, the GM MICs of posaconazole, amphotericin B and fluconazole in the CARD9 group were statistically higher and the GM MICs of terbinafine lower than those of undefined genetic background group. The FICI of the CARD9 group were higher than those of the undefined group in the combination of caspofungin plus amphotericin B and amphotericin B plus fluconazole, but lower than the undefined group in the combination of itraconazole plus terbinafine. CONCLUSIONS:The GM MICs for posaconazole, amphotericin B, and fluconazole were significantly elevated in the CARD9 group compared to the group with undefined chronic infections. For patients with refractory infections, conducting susceptibility testing before treatment can optimize the selection of the most effective therapeutic agent, and the combination therapy of caspofungin with amphotericin B or itraconazole may be considered the preferred treatment option.

ABSTRACTBackgroundMicrosporum canis, a dermatophyte commonly associated with pets, is a leading cause of severe tinea capitis. The increasing prevalence of antifungal resistance among dermatophytes poses a significant global health challenge.ObjectivesThis study aims to define the updated antifungal susceptibility profile of M. canis to enhance treatment strategies for dermatophyte infections.MethodsThis study analysed 348 M. canis isolates from mainland China for their susceptibility to 11 antifungal agents, following the CLSI M38‐A3 guidelines. Additionally, we investigated the susceptibility of M. canis to antifungal agents and analysed the correlation between in vitro drug susceptibility and clinical outcomes in 54 cases of tinea capitis.ResultsThe majority of strains showed low MICs to all 11 drugs. We described the upper limits of wild‐type (WT) minimal inhibitory concentrations (UL‐WT) for 10 of these agents; however, no clear resistance patterns were identified through MIC distribution analysis. Notably, fluconazole had the highest MICs among the tested classes, while the novel agent olorofim showed superior activity. Resistance was detected in two strains to griseofulvin (MIC 64 μg/mL), one to fluconazole (MIC 64 μg/mL) and two to terbinafine (MIC 16 μg/mL). Although azoles and terbinafine remain effective against M. canis. Clinical outcomes indicate that terbinafine may be less effective in treating M. canis infections.ConclusionEstablishing a clinical breakpoint for M. canis is urgently needed to improve treatment protocols. While azoles and griseofulvin are still recommended for M. canis infections, ongoing surveillance of dermatophyte species and their susceptibility to antifungal agents is crucial to guide treatment strategies.

Purpose Fungal rhinosinusitis is a significant and growing health concern in arid regions, with an increasing incidence over recent decades. Without timely and appropriate management, it can lead to severe complications, including potential intracranial spread. This study aims to establish efficient and rapid diagnostics for non-invasive fungal rhinosinusitis (FRS), addressing the challenge of its difficult-to-culture diagnosis. Methods Twenty-eight patients suspected of FRS were studied using endoscopic sinus surgery to obtain tissue samples for histopathology, direct microscopy, fungal culture, quantitative PCR (qPCR) and metagenomic next-generation sequencing (mNGS) detection. A patented qPCR targeting prevalent Aspergillus species was evaluated. Results The patient cohort had a male-to-female ratio of 9:14, with disease duration up to 50 years. Histopathologically, 23 out of 28 cases were positive. Fungal culture exhibited a sensitivity of 21.74%, with one false positive. qPCR and mNGS showed 100% sensitivity and specificity, with a 100% consistency rate for identification at the species level (23/23), and potential detection of cases with co-infections. The most common pathogen was A. flavus, followed by A. fumigatus and A. niger. Two cases involved mixed infections of A. fumigatus and A. flavus. Conclusion qPCR and mNGS proved effective in rapidly identifying fungi from fresh sinus tissue that are challenging to culture, surpassing conventional methods. However, further evaluation and optimization with a larger cohort of patients are necessary. Histopathology is still recommended to confirm the clinical significance of the detected fungal species.

Background: The fluorescent staining method with Calcofluor White (CFW) has been popularly used for the detection of fungi in clinical settings in recent years. However, seldom have new fluorochromes been invented to detect fungi. Purpose: To evaluate a new patented fluorochrome, Fluorescent Brightener 85 (FB 85), for detecting fungi in cultured fungi and superficial clinical specimens of fungal infectious disease. Methods: Initially, several pure fungi from different genera were cultured, stained with 0.1% FB 85, and observed with fluorescent microscopy. Subsequently, different superficial and invasive clinical specimens, including those from skin, nail debris, vaginal secretions, bronchoalveolar lavage fluid, and sputum, were stained with 0.1% FB 85 combined with 10% potassium hydroxide (KOH) and similarly observed with fluorescent microscopy. Last, Outpatients suspected of having superficial fungal infections were recruited. Samples were examined using both FB 85 fluorescent staining direct microscopy and 10% KOH direct microscopy to confirm diagnosis. Positive rates were compared, and statistical differences were analyzed. Results: Under fluorescent microscopy, for the cultured fungi, all the hyphae, conidia, conidiophores, septa, or bud scars were stained clearly, exhibiting strong blue fluorescence with high intensity that diminished slowly. Similarly, for both superficial and invasive clinical specimens, the strong, intense blue or blue-green fluorescence of the hyphae, pseudohyphae, budding, and septa was clearly stained, offering high contrast against the light blueish keratinocytes or epithelial cells in the background, and also diminished slowly. Finally, among 100 patients recruited, the positive rate of the FB 85 was 84%, while the KOH was 52%. Moreover, FB 85 method had a higher percentage of positive rate than KOH method, it had statistically significant difference ( χ2=23.53, P < 0.01). Conclusion: FB 85, the national invention patent by the author Yue, is a new and potentially perfect fluorescent reagent with excellent promise. It could be used for the diagnosis of fungal infections in clinical settings, as well as for observing the growth characteristics of fungi or the changes following antifungal drug therapy. For detecting fungi in superficial fungal infection samples in a clinical setting, the FB 85 method was more convenient, efficient, and accurate than the traditional KOH method.

Invasive aspergillosis (IA) is the most severe type of Aspergillus infection. Yunnan has developed agriculture, and the proportion of triazole-resistant A. fumigatus induced by triazole fungicides is much higher than that in other regions of China. Inhalation of triazole-resistant A. fumigatus is one of the main factors inducing IA. We gathered five strains of A. fumigatus from the sputum or bronchoalveolar lavage fluid (BALF) of patients with IA in Yunnan. Subsequent testing showed that all of these strains were resistant to triazoles and harboured mutations in the tandem repeat sequence of the cyp51A promoter region, suggesting that they may be triazole-resistant A. fumigatus present in the environment.