Fungal microbiome profiling in pulmonary sarcoidosis patients as revealed by target sequencing of biopsy, bronchoalveolar lavage fluid (BALF), and blood samples

Sarcoidosis is a granulomatous inflammatory disease of unknown etiology. Recent research has turned attention to the pulmonary fungiome as a potential contributor to its pathogenesis. Our aim was to profile the fungal biodiversity by sequencing of internal transcribed spacer (ITS) in lung biopsy, BALF, and blood samples from patients with sarcoidosis. Samples were collected from 15 sarcoidose patients according to standard clinical procedures. Additionally, a portion of each blood sample was grown on BHI medium with vitamin K (1 mg/mL) at 43 °C for 24 hours. Both cultured and non-cultured fractions were further analyzed. Total DNA was extracted and subjected to ITS-targeted sequencing. QIIME2 was used for taxonomic classification. Statistical analyses and visualizations were conducted in R and Python. A total of 2.06 million reads were obtained across all samples, with an average of approximately 31,000 reads per sample. Analysis revealed distinct fungal community structures across the different sample types. BALF samples were dominated by Penicillium (12%) and Candida (6%), with smaller contributions from Blumeria (5%), Amphinema (4%) and Rozellomycota (3%). In biopsy, a strong presence of Penicilium (12%), Amphinema (10%), and Homiactis (5%) was observed. All taxa presented in control were excluded. Cultured blood samples were enriched in Saccharomyces (12%), Blumeria (16%), Komagataella (7%), and Zoopagomycota (10%). Non-cultured blood samples displayed a distinct community structure, with elevated levels of environmental and slow-growing fungi such as Pseudoanungitea (8%), Salinomyces (7%), Rozellomycota (4%),  Ochrolechia (3%), and Thermomyces (3%). Washout samples had the highest relative abundance of Penicillium (18%) and were uniquely enriched with Nigrospora (8%). The findings suggest multiple environmental and endogenous origins of the blood and airway fungal communities. The prominence of opportunistic genera such as Candida, Saccharomyces, and Penicillium, may play a role in the pathogenesis of sarcoidosis. Future studies should explore the temporal dynamics of the diversity of pulmonary and blood fungiomes to clarify their contribution to disease onset, persistence, or resolution.