Beyond Fumigatus : a molecular portrait of clinical Aspergillus diversity, pathogenicity, and antifungal resistance

ABSTRACT Aspergillus infection poses a major clinical challenge, particularly in immunocompromised individuals, with invasive diseases associated with high mortality. While Aspergillus fumigatus remains the predominant species causing human infections, recent studies highlight the growing clinical significance of lesser-known and cryptic Aspergillus species, which often exhibit reduced susceptibility to standard antifungal therapies. In this study, we analyzed 196 clinical Aspergillus isolates from 107 patients treated at the NIH Clinical Center between 2019 and 2022. A total of 38 Aspergillus species across 11 taxonomic sections were identified, with non- fumigatus and cryptic species accounting for 77.1% of all isolates. The most frequently recovered species were A. fumigatus sensu stricto (22.9%), A. sydowii (8.7%), A. calidoustus (7.1%), A. nidulans (6.6%), A. tanneri (6.1%), and A. terreus (5.6%). Species-level identification was achieved in 43% of isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In contrast, PCR sequencing confirmed species identity in over 88% of isolates by targeting the internal transcribed spacer (ITS) region of rDNA, 81% using the β-tubulin gene, and 68% using the calmodulin gene. The most common underlying clinical conditions among patients were bronchiectasis (35%), chronic granulomatous disease (22%), and pulmonary non-tuberculous mycobacterial infection (17%). Out of 107 patients, eight died (8/107, 7.5%); six of these deaths occurred in patients with chronic granulomatous disease (CGD) and two in patients with RAG1 deficiency. Antifungal susceptibility testing showed that olorofim had the lowest minimal inhibitory concentrations across species. In contrast, the activity of triazoles and amphotericin B was variable, particularly against A. tanneri , A. calidoustus , and A. sydowii . This study presents one of the largest species-level data sets of Aspergillus isolates to date, underscoring the diversity, pathogenic potential, and resistance profiles of non- fumigatus species. Accurate species identification plays an important role in guiding appropriate antifungal therapy and improving clinical outcomes, although further studies are needed to elucidate its direct impact on treatment decisions.