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Introduction: Advances in diagnostic technologies, particularly Point-of-Care Diagnostics (POCDs), have revolutionized clinical practice by providing rapid, user-friendly, and affordable testing at or near the patient's location. POCDs have been increasingly introduced in medical mycology and hold promise to improve patient outcomes in a variety of important human fungal diseases., Areas Covered: This review focuses on validated POCDs, particularly lateral flow assays (LFAs), for various fungal diseases. Additionally, we discuss emerging innovative techniques such as body fluid analysis, imaging methods, loop-mediated isothermal amplification (LAMP), microfluidic systems, clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics, and the emerging role of artificial intelligence., Expert Opinion: Compact and user-friendly POCDs have been increasingly introduced in medical mycology, and some of these tests (e.g. Cryptococcus and Histoplasma antigen LFAs) have become mainstream diagnostics, while others, such as LFA in invasive aspergillosis show promise to become part of our routine diagnostic armamentarium. POCDs offer immense benefits such as timely and accurate diagnostic results, reduced patient discomfort, and lower healthcare costs and might contribute to antifungal stewardship. Integrated fluidics combined with microtechnology having multiplex capabilities will be pivotal in medical mycology.

Introduction: Pre-existent pools of coronavirus-specific or cross-reactive T cells were shown to shape the development of cellular and humoral immune responses after primary mRNA vaccination against SARS-CoV-2. However, the cellular determinants of responses to booster vaccination remain incompletely understood. Therefore, we phenotypically and functionally characterized spike antigen-specific T helper (Th) cells in healthy, immunocompetent individuals and correlated the results with cellular and humoral immune responses to BNT162b2 booster vaccination over a six-month period., Methods: Blood of 30 healthy healthcare workers was collected before, 1, 3, and 6 months after their 3rd BNT162b2 vaccination. Whole blood was stimulated with spike peptides and analyzed using flow cytometry, a 13-plex cytokine assay, and nCounter-based transcriptomics., Results: Spike-specific IgG levels at 1 month after booster vaccination correlated with pre-existing CD154+CD69+IFN-γ+CD4+ effector memory cells as well as spike-induced IL-2 and IL-17A secretion. Early post-booster (1-month) spike IgG levels (r=0.49), spike-induced IL‑2 (r=0.58), and spike-induced IFN‑γ release (r=0.43) correlated moderately with their respective long-term (6-month) responses. Sustained robust IgG responses were significantly associated with S-specific (CD69+±CD154+±IFN-γ+) Th-cell frequencies before booster vaccination (p=0.038), especially double/triple-positive type-1 Th cells. Furthermore, spike IgG levels, spike-induced IL‑2 release, and spike-induced IFN‑γ release after 6 months were significantly associated with increased IL‑2 & IL‑4, IP‑10 & MCP1, and IFN‑γ & IP‑10 levels at 1 month post-booster, respectively. On the transcriptional level, induction of pathways associated with both T-cell proliferation and antigen presentation was indicative of sustained spike-induced cytokine release and spike-specific IgG production 6 months post-booster. Using support vector machine models, pre-booster spike-specific T-cell frequencies and early post-booster cytokine responses predicted sustained (6-month) responses with F1 scores of 0.80-1.00., Discussion: In summary, spike-specific Th cells and T-cellular cytokine signatures present before BNT162b2 booster vaccination shape sustained adaptive cellular and humoral responses post-booster. Functional T-cell assays might facilitate early identification of potential non-responders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Page, Dennehy, Mueller, Girl, Loell, Buijze, Classen, Messmann, Roemmele, Hoffmann, Wurster and Fuchs.)

While mammalian models remain the gold standard to study invasive mycoses, mini-host invertebrate models have provided complementary platforms for explorative investigations of fungal pathogenesis, host-pathogen interplay, and antifungal therapy. Specifically, our group has established Toll-deficient Drosophila melanogaster flies as a facile and cost-effective model organism to study candidiasis, and we have recently expanded these studies to the emerging and frequently multidrug-resistant yeast pathogen Candida auris. Our proof-of-concept data suggest that fruit flies could hold a great promise for large-scale applications in antifungal drug discovery and the screening of C. auris (mutant) libraries with disparate pathogenic capacity. This chapter discusses the advantages and limitations of D. melanogaster to study C. auris candidiasis and provides a step-by-step guide for establishing and troubleshooting C. auris infection and antifungal treatment of Toll-deficient flies along with essential downstream readouts., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CD154+ mould-reactive T cells were proposed as a novel biomarker in the diagnosis of invasive mycoses. As PBMC-based protocols for flow cytometric quantification of these cells are logistically challenging and susceptible to preanalytic delays, this study evaluated and optimized a whole blood-based method for the detection of mould-reactive T cells. Blood collection tubes containing costimulatory antibodies and Aspergillus fumigatus mycelial lysates were inoculated with heparinized whole blood from healthy adults, and detection rates of CD154+/CD4+A. fumigatus reactive T cells were compared with PBMC-based detection using samples from the same donors. In contrast to the PBMC-based method, double costimulation with αCD28 and αCD49d was crucial for reliable whole blood stimulation. Optimizing stimulation schemes for both matrixes, significantly higher specific T-cell detection rates were achieved by the whole blood-based method, whereas the unspecific background stimulation remained low. MHC II-dependent CD154+ upregulation was demonstrated for both matrixes. Excellent correlation and reproducible conversion factors between whole blood and PBMC-based results were observed. Using frozen ready-to-use test tubes containing costimulatory antibodies and lysates, detection rates of specific T cells were comparable to freshly prepared blood collection tubes. The optimized whole blood-based protocol was also used to detect Rhizopus arrhizus and Rhizomucor pusillus reactive T cells, resulting in 1.5- to 2.7-fold higher detection rates compared with PBMC-based measurement. In summary, the whole blood protocol is a robust, highly sensitive, and cost-effective method for mould-reactive T-cell quantification, allowing for point-of-care sample stimulation and contributing to better assay standardization in multi-centre evaluation of mould reactive T-cell quantification., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell ® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus , and Cunninghamella bertholletiae . Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell ® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.