High-Volume Culture Improves Diagnostic Yield of Aspergillus Species From Sputum

Objectives: Sputum is a commonly collected specimen for suspected respiratory fungal infections due to its non-invasive nature and diagnostic value. However, conventional culture at 29°C (CC29) often fails to detect filamentous moulds. To improve recovery, we applied a high-volume culture incubated at 37°C (HVC37) and compared its diagnostic performance with CC29, with particular focus on Aspergillus species and azole-resistant isolates. Methods: Between October 2022 and June 2025, a total of 1546 sputum samples from 793 patients, including 219 samples from 67 patients with chronic pulmonary aspergillosis (CPA), were cultured in parallel using HVC37 (0.5–1 mL inoculum) and CC29 (1–10 μL inoculum) on Sabouraud agar for 7 days. Positivity rates for moulds and Aspergillus species were calculated with 95% confidence intervals. Antifungal susceptibility testing followed EUCAST standards. Results: High-volume culture incubated at 37°C (HVC37) markedly outperformed CC29 for detection of both moulds and Aspergillus species. Across all sputum samples, HVC37 achieved a mould positivity rate of 41.6% (95% CI 39.2%–44.1%) versus 27.7% (95% CI 25.5%–30.0%) with CC29. The improvement was most evident for Aspergillus detection: 37.0% (95% CI 34.7%–39.5%) for HVC37 compared with 11.5% (95% CI 10.0%–13.2%) for CC29, an absolute increase of 25.5%. Both methods together yielded the highest overall detection rates for moulds (54.0%) and Aspergillus species (42.1%). In patients with CPA, HVC37 detected moulds in 53.0% of samples and Aspergillus species in 45.2%, compared with 30.6% and 15.5%, respectively, for CC29. Importantly, of 46 azole-resistant A. fumigatus isolates recovered, HVC37 detected 42 (91.3%), whereas CC29 detected only 20 (43.5%). CC29 mainly identified environmental moulds such as Penicillium species, not detected by HVC37. Conclusions: High-volume culture at 37°C substantially improves the detection of filamentous moulds in sputum, especially Aspergillus species and clinically relevant azole-resistant A. fumigatus. Incorporating HVC37 into routine mycology workflows can significantly enhance fungal diagnosis and antifungal resistance surveillance.