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Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

The rise of microbial pathogens refractory to conventional antibiotics represents one of the most urgent and global public health concerns for the 21st century. Emergence of Candida auris isolates and the persistence of invasive mold infections that resist existing treatment and cause severe illness has underscored the threat of drug-resistant fungal infections. To meet these growing challenges, mechanistically novel agents and strategies are needed that surpass the conventional fungistatic or fungicidal drug actions. Host defense peptides have long been misunderstood as indiscriminant membrane detergents. However, evidence gathered over the past decade clearly points to their sophisticated and selective mechanisms of action, including exploiting regulated cell death pathways of their target pathogens. Such peptides perturb transmembrane potential and mitochondrial energetics, inducing phosphatidylserine accessibility and metacaspase activation in fungi. These mechanisms are often multimodal, affording target pathogens fewer resistance options as compared to traditional small molecule drugs. Here, recent advances in the field are examined regarding regulated cell death subroutines as potential therapeutic targets for innovative anti-infective peptides against pathogenic fungi. Furthering knowledge of protective host defense peptide interactions with target pathogens is key to advancing and applying novel prophylactic and therapeutic countermeasures to fungal resistance and pathogenesis.

Influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) affect about 15% of critically ill patients with influenza or COVID-19, respectively. These viral–fungal coinfections are difficult to diagnose and are associated with increased mortality, but data on their pathophysiology are scarce. We aimed to explore the role of lung epithelial and myeloid innate immunity in patients with IAPA or CAPA. In this observational study, we retrospectively recruited patients who had been admitted to the intensive care unit (ICU) of University Hospitals Leuven, Belgium, requiring non-invasive or invasive ventilation because of severe influenza or COVID-19, with or without aspergillosis, between Jan 1, 2011, and March 31, 2021, whose bronchoalveolar lavage samples were available at the hospital biobank. Additionally, biobanked in vivo tracheobronchial biopsy samples from patients with IAPA or CAPA and invasive Aspergillus tracheobronchitis admitted to ICUs requiring invasive ventilation between the same dates were collected from University Hospitals Leuven, Hospital Network Antwerp (Belgium), and Amiens-Picardie University Hospital (France). We did nCounter gene expression analysis of 755 genes linked to myeloid innate immunity and protein analysis of 47 cytokines, chemokines, and growth factors on the bronchoalveolar lavage samples. Gene expression data were used to infer cell fractions by use of CIBERSORTx, to perform hypergeometric enrichment pathway analysis and gene set enrichment analysis, and to calculate pathway module scores for the IL-1β, TNF-α, type I IFN, and type II IFN (IFNγ) pathways. We did RNAScope targeting influenza virus or SARS-CoV-2 RNA and GeoMx spatial transcriptomics on the tracheobronchial biopsy samples. Biobanked bronchoalveolar lavage samples were retrieved from 166 eligible patients, of whom 40 had IAPA, 52 had influenza without aspergillosis, 33 had CAPA, and 41 had COVID-19 without aspergillosis. We did nCounter gene expression analysis on bronchoalveolar lavage samples from 134 patients, protein analysis on samples from 162 patients, and both types of analysis on samples from 130 patients. We performed RNAScope and spatial transcriptomics on the tracheobronchial biopsy samples from two patients with IAPA plus invasive Aspergillus tracheobronchitis and two patients with CAPA plus invasive Aspergillus tracheobronchitis. We observed a downregulation of genes associated with antifungal effector functions in patients with IAPA and, to a lesser extent, in patients with CAPA. We found a downregulated expression of several genes encoding proteins with functions in the opsonisation, recognition, and killing of conidia in patients with IAPA versus influenza only and in patients with CAPA versus COVID-19 only. Several genes related to LC3-associated phagocytosis, autophagy, or both were differentially expressed. Patients with CAPA had significantly lower neutrophil cell fractions than did patients with COVID-19 only. Patients with IAPA or CAPA had downregulated IFNγ signalling compared with patients with influenza only or COVID-19 only, respectively. The concentrations of several fibrosis-related growth factors were significantly elevated in the bronchoalveolar lavage fluid from patients with IAPA versus influenza only and from patients with CAPA versus COVID-19 only. In one patient with CAPA, we visualised an active or very recent SARS-CoV-2 infection disrupting the epithelial barrier, facilitating tissue-invasive aspergillosis. Our results reveal a three-level breach in antifungal immunity in IAPA and CAPA, affecting the integrity of the epithelial barrier, the capacity to phagocytise and kill Aspergillus spores, and the ability to destroy Aspergillus hyphae, which is mainly mediated by neutrophils. The potential of adjuvant IFNγ in the treatment of IAPA and CAPA should be investigated. Research Foundation Flanders, Coronafonds, the Max Planck Society, the Fundação para a Ciência e a Tecnologia , the European Regional Development Fund, "la Caixa" Foundation, and Horizon 2020. [ABSTRACT FROM AUTHOR]