Histology-Correlated FTIR Chemical Imaging of Fungal Infection-Associated Tissue Compartments in Human Skin: A...

Background/Objectives: Fungal skin infections are commonly assessed using clinical examination and conventional histopathology, including hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Grocott methenamine silver (GMS) staining. However, these methods provide limited spatially resolved biochemical information. This proof-of-concept study investigated whether Fourier transform infrared (FTIR) chemical imaging can identify biochemical patterns associated with histologically defined fungal infection-associated tissue compartments in human skin. Methods: Archived formalin-fixed, paraffin-embedded skin samples with histological evidence of fungal infection were investigated. The study group comprised 19 patients, of whom 12 fulfilled the histological and technical eligibility criteria for quantitative FTIR analysis. These 12 independent biological cases yielded 46 histologically defined regions of interest (ROIs), comprising 16 fungal infection-associated ROIs, 14 keratosis/keratinised tissue ROIs, and 16 vital epidermis ROIs. ROI assignment was guided by corresponding HE-, PAS-, and GMS-stained sections. Results: Fungal infection-associated tissue compartments showed partially distinct spectral characteristics compared with vital epidermis and keratinised tissue. The most prominent exploratory differences occurred within the 900-1300 cm^-1 fingerprint region. Case-level statistical analysis showed significant differences between fungal infection-associated tissue and vital epidermis at approximately 1185 and 1240 cm^-1 after false discovery rate correction, whereas substantial overlap with keratinised tissue remained. Case-level PCA retained tissue-associated spectral structure after biological aggregation, although fungal infection-associated and keratinised tissue showed partial overlap. Unsupervised clustering further demonstrated spatially coherent spectral compartments corresponding to histologically identifiable tissue structures. Conclusions: FTIR chemical imaging may complement conventional histopathology by providing label-free, spatially resolved biochemical information on fungal infection-associated tissue compartments. Because fungal elements are embedded within surrounding keratinised and epithelial tissue, the observed spectral characteristics should be interpreted as exploratory infection-associated tissue signatures rather than fungal-specific diagnostic biomarkers. Larger independent studies with case-wise validation are required before diagnostic application can be considered.