Introduction: To compare EORTC/MSGERC and BM-AspICU diagnostic criteria for invasive aspergillosis and analyse clinical, mycological, and radiological features affecting patient outcomes. Materials and Methods: We conducted a retrospective study on 56 invasive aspergillosis patients who met inclusion criteria (age >18 years and confirmed diagnosis via histopathology, cultures, or galactomannan). Patients were categorized using EORTC/MSGERC and BM-AspICU criteria. Clinical, microbiological, radiological data, and outcomes were analysed. Results: of 56 patients, 47 had invasive pulmonary aspergillosis, 7 had invasive rhinosinusitis, 1 had disseminated, and 1 had cerebral aspergillosis. Mean age was 51.5 years, with 75% (42) males. Aspergillus fumigatus was common species (47.3%) isolated. Computed tomography scans of invasive pulmonary aspergillosis often showed nonspecific infiltrates (31.9%). Voriconazole was most used antifungal (80.4%). Mortality was 23.2% (n=13) in total and in intensive care unit patients with invasive pulmonary aspergillosis was 46.1% (n=12). 75% (9/12) of deceased patients initially classified as pulmonary colonizers by EORTC/MSGERC were reclassified as probable (n=3) or possible (n=6) invasive aspergillosis cases by BM-AspICU, difference was statistically significant (p=0.019). Intensive care unit admission was 55.3% (median stay: 8 days), higher in viral pneumonia (p = 0.003). Univariate analysis of invasive pulmonary aspergillosis patients showed significant mortality correlations with shock (p=0.001), acute kidney injury (p=0.003), invasive mechanical ventilation (p=0.001) and intensive care unit stay (p=0.026). Multivariate analysis identified shock as an independent predictor of mortality. Conclusion: In our study, BM-AspICU criteria demonstrated a stronger correlation with mortality compared to EORTC/MSGERC criteria. Morbidity and mortality were associated with viral pneumonia, shock, acute kidney injury, invasive mechanical ventilation and intensive care unit stay.

Human-pathogenic molds produce various natural products (xenobiotics) that interact with human host proteins. Here, a sequence- and structure-based virtual screening pipeline for protein-xenobiotic interactions was established. Verification tests run on Methylene Blue and Fumagillin identified the known top interaction targets in both cases. The pipeline was then applied to 1,8-dihydroxynaphthalene (DHN)-melanin, an Aspergillus fumigatus spore pigment and virulence determinant. Our sequence-structure aggregated scoring for DHN-melanin-interacting proteins included protein localization, evolution, and function. Experimental data validated our identified candidates and support ranking accuracy. Conserved targets were further assessed: for their possible participation in the macrophage response to A. fumigatus (using gene ontology terms). Lower-scoring targets, such as glucosidase alpha (GAA; experimental data in this paper) and sentrin-specific protease 8 (SENP8), were shown not to bind DHN-melanin by molecular dynamics (MD) simulations as well as experiments, supporting their lower pipeline scores. The best DHN-melanin interacting candidates were validated by docking and molecular dynamics simulations, resulting in the identification of the most likely DHN-melanin-interacting proteins in human immune and epithelial cells. The pipeline identified the following previously validated DHN-melanin interaction targets with top score: C-type lectin receptor MelLec (CLEC1A) and surfactant protein-D (SP-D). In addition, two novel targets share the same top score, the aromatic hydrocarbon receptor (AHR) and methionine aminopeptidase 2 (METAP2), the latter of which was experimentally validated.

Invasive aspergillosis (IA) remains a major cause of morbidity and mortality in immunocompromised individuals, with an expanding spectrum of at-risk populations, particularly in intensive care settings. Despite advances in antifungal pharmacotherapy, treatment outcomes remain suboptimal, and the rise of antifungal resistance highlights the need for adjunctive therapy that leverages host immune mechanisms. The pathogenesis of IA is primarily driven by impaired or dysregulated immune responses to Aspergillus species, mainly Aspergillus fumigatus. Host defence involves a complex interplay of innate and adaptive, cellular and humoral immune components. However, this response is not only intricate but also heterogenous, challenging the effectiveness of uniform treatment approaches. This review aims to examine the diverse roles of immune components in host-Aspergillus interactions and to explore how these insights are currently informing the development of novel immune-targeted therapies. A thorough analysis of recent and relevant peer-reviewed literature was conducted to synthesize current understanding of Aspergillus-host interactions and the emerging therapeutic strategies derived from these insights. Following a brief overview of the immunopathogenesis of IA, we integrate findings from both preclinical models and clinical studies to evaluate the potential of immune-based therapies as adjuncts to traditional antifungal treatment. Emphasis is placed on the promise of personalized approaches tailored to individual immune profiles. We also address existing knowledge gaps and outline key directions for future research and clinical application. A deeper understanding of host immunity in IA paves the way for more precise, effective, and personalized therapeutic strategies, with the potential to significantly improve outcomes in vulnerable patient populations. Although still in its early stages, targeted immunotherapy represents an emerging and promising perspective in infectious diseases management. Insights gained from IA may also inform the development of immune-based interventions for other opportunistic infections.

Ubiquinone-based gene mutation and protein compactness of CoQ5 may contribute to a novel caspofungin resistance mode in Aspergillus flavus from pulmonary aspergillosis.

Rapid antifungal susceptibility testing for Aspergillus fumigatus using a loop-mediated isothermal amplification method.

Ligand-Based Design of Novel Thiazole-Quinazolinone Hybrids with Dual Antimicrobial and Anti-Virulence Activity Targeting Staphylococcal Sortase A.

Decoding azole resistance mechanisms and pathogenicity in Aspergillus section Fumigati through genomic analysis.

Avian aspergillosis, caused by Aspergillus fumigatus (Af), lacks sensitive antemortem diagnostics. Existing microbial cell-free DNA (cfDNA) tests are prone to contamination and require a high pathogen load. We hypothesized that infection-induced tissue damage in chickens creates differentially methylated regions (DMRs) in host cfDNA, enabling machine learning (ML) diagnostics. Serum cfDNA samples (n = 124) were obtained from broiler chickens (n = 76) with Af and non-Af infections (Escherichia coli or Gallibacterium anatis) and controls. Oxford Nanopore sequencing enabled DMR detection and ML training. Performance was evaluated using an independent set (n = 49) and 10-repeat Monte Carlo cross-validation (CV) (n = 490 evaluations per test) as quality control. A High Accuracy test (93 DMRs, neural network) achieved 98.0% accuracy (sensitivity 95%, specificity 100%, AUC 0.974, PR-AUC 0.928) in the independent set, with CV accuracy 92.0% [95% CI: 89.7%-94.4%]. A Fast test (35 DMRs, SVM) achieved 81.6% accuracy and CV accuracy 79.6% [74.9%-84.3%]. An In Situ test (5 DMRs, random forest) designed for field deployment achieved 71.4% accuracy and CV accuracy 62.9% [58.7%-67.0%]. Stratified CV accuracy showed 84.6% [65.1%-95.6%] correct classifications for E. coli and 100% [80.5%-100%] for G. anatis. Markers showed high bootstrap stability and predominantly overlapped EMARs and enhancers. In conclusion, we present MethylSense (https://github.com/markusdrag/MethylSense), an automated open-source software. The High Accuracy test achieved 92.0% [89.7%-94.4%] CV accuracy (CV sensitivity 94.5% [91.4%-97.6%], CV specificity 90.3% [87.8%-92.9%]). While validated in chickens, MethylSense is adaptable to other species and pathogens, offering scalable, contamination-resilient diagnostics for veterinary and conservation applications.IMPORTANCEMethylSense is an automated software for training machine learning diagnostics using differentially methylated regions (DMRs) in cell-free DNA from Oxford Nanopore sequencing. We applied MethylSense to develop three Aspergillus fumigatus tests for chickens, each optimized for different clinical scenarios. The High Accuracy test (93 DMRs, neural network) demonstrated 98.0% accuracy, in a blinded test set (n = 49) with sensitivity 95%, specificity 100%, ROC-AUC 0.974, and PR-AUC 0.928. Stratified 10-repeat Monte Carlo cross-validation (n = 490) showed correct classifications of 84.6% [CI: 65.1%-95.6%] Escherichia coli and 100% [80.5%-100%] Gallibacterium anatis infected specificity samples. A Fast test for rapid <1 h sequencing (35 DMRs, support vector machine) achieved 81.6% accuracy (sensitivity 80%, specificity 82.8%). An In Situ test (5 DMRs, random forest) for field deployment via methylation-specific PCR achieved 71.4% accuracy (sensitivity 45%, specificity 89.7%). Bootstrap analysis demonstrated exceptional marker stability (80.6%-100%) with minimal batch effects, confirming robust host-based diagnostics.

Aspergillus fumigatus resistance to triazole antifungals poses an increasing global health concern. Moreover, the cross-resistance between azole antifungal agents used in clinical settings and those applied in agriculture has become an important emerging issue. In this study, we investigated the five environmental A. fumigatus strains showing reduced susceptibility to tebuconazole. Fungal strains were recovered from air samples collected around the homes of two patients with suspected aspergillosis caused by resistant isolates. Species identification was performed by sequencing the β-tubulin gene, and minimum inhibitory concentrations were determined by broth microdilution. The cyp51A gene was sequenced to detect mutations, and CRISPR-Cas9 genome editing was employed to investigate their influence on susceptibility patterns. Microsatellite genotyping was performed to assess genetic variability, followed by whole genome sequencing and single nucleotide polymorphism analysis. The environmental strains presented the same cyp51A genotype characterised by the M172V substitution and silent mutations. Microsatellite genotyping and whole genome sequencing confirmed that the strains were clonal. Functional validation demonstrated that the M172V and silent mutations partially contribute to reduced susceptibility to tebuconazole but are not the main mechanism of resistance involved. Analysis of polymorphisms in genes other than cyp51A revealed no resistance-conferring mutations. The findings described herein suggest the possibility of local clonal dissemination of environmental strains under selective pressure from agricultural azoles in a major agribusiness region of the Midwest of Brazil. This study highlights the silent spread of potentially resistant genotypes in urban areas and reinforces the need for environmental surveillance and expanded genomic monitoring in South America.

Historical manuscripts are invaluable cultural artifacts whose preservation is crucial for safeguarding human heritage. This study investigated fungal contamination in twenty-five deteriorated paper-based historical manuscripts from the Istanbul University Virtual Library. Samples were collected using both swab and membrane filter methods, and fungal isolates were cultivated on dichloran glycerol 18 agar and malt extract agar. Cellulase activity was screened among isolates, followed by morphological identification. A total of 199 fungal isolates were obtained, 93.46% of which were cellulolytic, and their enzymatic indices ranged from 1.16 to 4.00. Swab sampling yielded significantly higher colony counts than membrane filtration, although no significant difference was found between media. The most frequently encountered species were Penicillium chrysogenum and Aspergillus fumigatus. The findings emphasize the necessity of using complementary sampling techniques and culture media to assess microbial diversity and enzymatic activity in historical documents, thereby informing more effective conservation strategies.

Dominance of Bacillus species on conventional agar often hampers the recovery of diverse soil bacteria ("Bacillus dominance"). To address this, we developed an agar medium containing leaf mold extract (LME) and evaluated its effectiveness for soil microorganism isolation compared to conventional yeast malt extract agar (YME agar). The 16S rRNA gene sequencing revealed that YME agar cultures were dominated by Bacillus-related bacteria, while LME agar enabled the growth of a broader range of taxa, especially actinomycetes like Streptomyces. LME agar supported higher bacterial diversity even in samples with high Bacillus abundance. Some LME isolates had very low 16S rRNA gene similarity to known species or could not be amplified with universal primers, suggesting potentially novel taxa. One unidentified isolate proliferated only in liquid LME, indicating a unique nutrient requirement. We further screened LME-derived isolates (569 strains) for therapeutic activity using an insect (the silkworm, Bombyx mori) model infected with three pathogenic fungi (Rhizopus arrhizus, Aspergillus fumigatus, Candida albicans). Distinct extracts showed significant efficacy in each infection model, and the overall hit rate was 6.2%. Notably, the extract of the rare species Ancylobacter sonchi showed marked efficacy against R. arrhizus infection, underscoring LME agar's potential for accessing novel bioresources. These results suggest that LME agar suppresses Bacillus dominance and expands access to diverse, rare, and previously uncultured soil bacteria, providing a powerful platform for microbial library construction and novel drug discovery. KEY POINTS: • LME agar prevents Bacillus dominance, enabling recovery of diverse microbial colonies. • LME agar accesses rarer and more diverse microbiota than nutrient-rich media. • A strain from LME agar showed therapeutic activity in silkworms infected withR. arrhizus.

ABSTRACT Periodic surveillance of antifungal susceptibility among Aspergillus isolates is essential to guide effective treatment. This multicenter study analyzed 550 clinical Aspergillus isolates collected in Taiwan from 2021 to 2023, primarily from the respiratory tract (69.6%) and ear (12.9%). Calmodulin-based sequencing identified 24 species across six Aspergillus sections: Flavi (28.7%), Fumigati (28.5%), Nigri (21.6%), Terrei (15.8%), Nidulantes (5.1%), and Circumdati (0.2%). The major sections were represented by Aspergillus fumigatus (99.4%), Aspergillus flavus (91.8%), and Aspergillus terreus (100%), while section Nigri comprised nine species, with Aspergillus welwitschiae (57.1%) predominant. Using Clinical Laboratory Standards Institute (CLSI) M38-A3 protocols, reduced susceptibility to amphotericin B (MIC &gt; 1 µg/mL) was observed in A. flavus (8.3%), A. terreus (22.7%), and section Nidulantes (32.1%). Acquired voriconazole resistance was found in 5.8% (9/156) of A. fumigatus isolates, including eight with cyp51A mutations (3 TR 34 /L98H, 4 TR 34 /L98H/S297T/F495I, and 1 TR 46 /Y121F/T289A). Voriconazole non-wild-type phenotypes were identified in 2.1% (3/158) of A. flavus isolates, including one carrying a novel P214L mutation (orthologous to A. fumigatus P216L). Members of the Aspergillus niger clade exhibited reduced susceptibility to itraconazole and isavuconazole. Anidulafungin, along with novel antifungals including orolofim, manogepix, and rezafungin, demonstrated broad activity across Aspergillus species, including those with intrinsic or acquired azole resistance. Ibrexafungerp also showed efficacy against azole-resistant A. fumigatus and A. flavus . In contrast, opelconazole exhibited limited activity against A. flavus , the A. niger clade, Aspergillus sydowii , and voriconazole-resistant A. fumigatus . These findings underscore the need for species-level identification, susceptibility testing of causative isolates, and continued surveillance to detect emerging resistance and support the use of novel non-azole antifungals for azole-resistant aspergillosis. IMPORTANCE Timely and effective antifungal therapy is essential for aspergillosis. This multicenter surveillance study provides comprehensive insights into the species distribution and antifungal susceptibility of 550 clinical Aspergillus isolates in Taiwan, with intrinsic reduced susceptibility to amphotericin B, itraconazole, or isavuconazole noted in certain species. Regarding acquired resistance, a novel cyp51A mutation, P214L, was identified in an azole-resistant Aspergillus flavus , orthologous to Aspergillus fumigatus P216L. Recovery of azole-resistant A. fumigatus harboring TR34/L98H or TR46/Y121F/T289A mutations remains a concern and emphasizes the need for antifungal stewardship in the environment. Novel antifungals, including orolofim, manogepix, rezafungin, and ibrexafungerp, demonstrated broad activity across Aspergillus species, including resistant isolates. Nevertheless, the inhaled agent opelconazole exhibited limited activity against A. flavus regardless of voriconazole susceptibility and against other species showing reduced susceptibility to itraconazole. These findings highlight the importance of species-level identification, susceptibility testing, and continued surveillance and support the use of novel antifungals for aspergillosis.

Fungal infections in shrimp are one of the main challenges in shrimp farming activities that have the potential to reduce productivity levels and crop quality. Fungi are known as opportunistic microorganisms that can cause serious problems, especially when pond environmental conditions are not supportive. Fungal attacks generally occur when shrimp experience physiological stress due to temperature fluctuations, changes in salinity, decreased water quality, or excessively high stocking densities. This study was conducted to identify and characterize the types of fungi associated with shrimp ponds in three main cultivation areas in East Java, namely Banyuwangi, Jember, and Gresik. The fungal isolation process was carried out using Sabouraud Dextrose Agar (SDA) media and incubated at 27°C. Morphological identification was carried out by observing the characteristics of colonies, the shape and color of hyphae, and the structure of conidia. The results of the observations obtained were 6 fungal isolates divided into two main groups, namely yeast including Candida sp., Rhodotorula sp., and Brettanomyces sp. The most commonly found mold species were Aspergillus flavus, Aspergillus fumigatus, and Aspergillus niger. The results of this study demonstrate the potential of fungal probiotics in shrimp farming ecosystems and serve as a basis for developing disease prevention strategies for whiteleg shrimp in East Java's shrimp ponds.

Invasive pulmonary aspergillosis (IPA) poses a serious threat to immunocompromised hosts, with limited therapeutic options highlighting the need for novel strategies. Coptis chinensis Franch. (CCF), a traditional Chinese herb containing antimicrobial alkaloids like berberine, was investigated for its therapeutic efficacy and immunological effects in a murine IPA model. Immunosuppressed female KM mice infected with Aspergillus fumigatus AF293 were treated with CCF or amphotericin B (AmB). CCF significantly improved survival, reduced fungal burden, and alleviated lung pathology, without inducing hepatotoxicity or nephrotoxicity. Transcriptomic profiling revealed a time-dependent immune response. Complement-related pathways were enriched at 2 days post-infection, whereas neutrophil recruitment and NET-related pathways became more prominent by day 4. Hub gene analysis identified Syk, Rac2, Ncf1, and Cybb as key nodes associated with the NADPH oxidase complex. Western blot and inhibitor experiments further supported the involvement of this pathway in CCF-mediated protection. Additionally, 16S rDNA sequencing indicated enrichment of Clostridium species in the gut microbiota of CCF-treated mice, which was positively correlated with the expression of NADPH oxidase-related genes, suggesting a potential gut-lung association. In conclusion, these findings support the antifungal efficacy of CCF in IPA and suggest that its protective effects may involve coordinated changes in complement-related responses, NADPH oxidase-associated neutrophil activity, and gut microbiota composition.

Aspergillus fumigatus is a leading cause of invasive fungal disease in humans and is classified as a critical priority threat by the World Health Organization. Triazole antifungals remain the cornerstone of therapy, yet their effectiveness is steadily being eroded by the continuous rise in drug resistance. Most resistance mechanisms trace back to mutations in Cyp51A, spawning well-defined genotypes such as TR34/L98H and TR46/Y121F/T289A. However, the Cyp51A genotype-phenotype landscape in A. fumigatus is far from straightforward. Isolates that share an identical TR genotype can display strikingly divergent susceptibility profiles, and mutational hotspots in Cyp51A, such as G54, M220 and G448, are linked to varying resistances, challenging assumptions about predictable resistance behavior. Complicating matters further, an expanding array of resistance mechanisms, independent of Cyp51A, is now being uncovered. This review summarizes the current state of knowledge on azole resistance in A. fumigatus, dissecting the intricate genotype-phenotype relationships, spotlighting emerging non-Cyp51A pathways and outlining future strategies to enhance the detection and clinical management of antifungal resistance.

Background: Zanthoxylum gilletii, an important African medicinal plant, is widely used in traditional medicine for treating various ailments due to its rich phytochemical composition. However, despite its extensive ethnomedicinal applications, the specific bioactive compounds responsible for its antimicrobial properties remain insufficiently characterized. Aims: The study aims to isolate and characterise bioactive compounds from Zanthoxylum gilletii stem bark and evaluate their antimicrobial activity against selected bacterial and fungal pathogens. Methodology: Zanthoxylum gilletii stem bark was collected from Mabira Forest Reserve (0°23′54″N 33°0′59″E), Buikwe District, Uganda on 17th June 2022. The experiments were performed at the Department of Chemistry, Kyambogo University, between June 2022 and July 2023. Stem barks were shade dried, powdered, and extracted using Maceration technique with a mixture of methanol and dichloromethane (ratio 1:1) solvent. The extract was subjected to open column chromatography and identified using NMR spectroscopy and compared with literature information. Antimicrobial activity of crude extract and also the isolated compounds were assessed via disk diffusion against five bacterial and two fungal strains. Results: In this study, four known compounds: lupeol (1), stigmasterol (2), α-amyrin cinnamate (3) and α-amyrin acetate (4) were isolated from stem bark extracts of Zanthoxylum gilletii. This is the first-time compound 4 is reported in Zanthoxylum genus. The compounds had antimicrobial activities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Candida albicans and Aspergillus fumigatus. The extract and the compounds displayed inhibition against the microorganisms with diameters measuring 3.0±0.0 mm to 19.0±0.0 mm. The minimal inhibitory concentrations (MICs and MFCs) for active strains ranged from 6.25 to 150 mg/mL. Conclusion: The observed activities support the traditional use of this plant in treating various ailments by Ugandan communities.

Falcon plasma proteomics to improve avian aspergillosis diagnosis.

ABSTRACT Water borne diseases have become serious public health concern in developing countries due to contaminated water. In Pakistan most of the diseases emerge due to water borne pathogens and the fatality rate is high. Water quality can be enhanced by household water treatment by means of silver nanoparticle impregnated clay‐agrowaste nanofilters. In this study, low‐cost technology was used to treat water at household level for 12 months, therefore AgNPs were generated using Aspergillus fumigatus (BTCC10). Before treatment, three parameters were optimized, that is biomass weight (7, 13 and 16 g), NaCl (20%) and sunlight exposure. AgNPs formed were characterized by UV–vis spectrophotometer, Zeta sizer, scanning electron microscopy and Fourier transform infrared (FTIR). After optimization, it was revealed that 13 g biomass had the smallest average size AgNPs (91.7 nm), along with zeta potential of −38.9 mV and polydispersity index (PDI) 0.19 whereas rest of the parameters experienced increase in size. Correspondingly, the functional groups were identified via FTIR as C─H, O─H, C═C, C═O, and N─H acted as capping agents. Later, silver impregnated clay pot filter (CPF), bagasse clay pot filter, and sawdust clay pot filter (SCPF) were prepared. AgNPs‐coated SCPF produced cleaner water as compared to others with reduction by 3 to 4‐fold in contrast to control and proved to be an efficient and sustainable approach for water purification. Additionally, it will also be suitable for areas where water is scarce and unfit for human consumption. Antibacterial activity was also performed against multi‐drug resistant bacterial strains and maximum zones of inhibition (ZOI) for AgNPs against Salmonella typhi was obtained at 16 ± 0.20 d mm whereas Staphylococcus aureus attained ZOI at 14 ± 1.00 b mm with 13 g biomass.

RTA-like proteins regulate azole susceptibility by affecting the expression of the oxidoreductase gene oxrA in Aspergillus fumigatus.

Natural killer (NK) cells play a key role in combating invasive mycoses, with surface proteins crucial for immune cell-target interactions. Here, we investigate the role of DNAX accessory molecule 1 (DNAM-1; CD226) in the recognition of Aspergillus fumigatus and Candida albicans hyphae. Super-resolution microscopy reveals a uniform distribution of DNAM-1 on naïve NK cells that is maintained upon contact with fungal hyphae. However, soluble DNAM-1 binds to the fungal cell wall and blocking DNAM-1 reduces antifungal activity in colony-forming assays. Furthermore, in silico domain-domain interaction analysis identifies several fungal proteins as putative DNAM-1 targets, including OpsB in A. fumigatus and Sap10 in C. albicans. High-affinity binding between Sap10 and DNAM-1 is predicted computationally and confirmed experimentally by co-immunoprecipitation and fluorescence correlation spectroscopy. Microscopic analysis further demonstrates that Sap10 binds to primary NK cells but not to DNAM-1-deficient NK cells. Sap10 binding induced NK-cell activation, as evidenced by increased CD69 expression and elevated perforin and CCL3 secretion. These findings identify Sap10 as a fungal ligand of DNAM-1 and reveal a mechanism by which NK cells recognize fungal pathogens.