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

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.

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.

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.

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.