A tiered tissue-based framework for diagnosing invasive fungal diseases: Integrating histopathology with targeted and metagenomic sequencing.

Invasive fungal diseases remain diagnostically challenging because clinical manifestations, radiological findings, biomarkers, culture, histopathology, and molecular assays each provide incomplete evidence. Histopathology is essential for demonstrating fungal elements within damaged or invaded tissue, but morphology alone often cannot identify fungi to the genus or species level. Targeted fungal PCR and sequencing can improve etiological assignment when fungal elements are visible in tissue, whereas metagenomic next-generation sequencing may add value in culture-negative, mixed, rare, or morphologically ambiguous cases. However, broad sequencing should be used selectively because of cost, host-background interference, contamination risk, database limitations, and uncertain tissue-specific reporting thresholds. We propose a pathology-driven tiered framework that begins with clinical triage and specimen allocation, proceeds through histopathology and special stains, applies morphology-directed targeted molecular testing, and reserves metagenomic sequencing for selected unresolved cases. This framework complements existing guidelines and laboratory standards, supports integrated reporting, and promotes diagnostic stewardship in tissue-based fungal diagnosis.