Beyond detection: quantitative interpretation of Aspergillus-positive bronchoalveolar lavage fluid metagenomic next-generation sequencing for diagnostic stratification and prediction of respiratory deterioration.

BACKGROUND

The increasing use of bronchoalveolar lavage fluid (BALF) metagenomic next-generation sequencing (mNGS) has substantially improved the detection of Aspergillus species in patients with suspected pulmonary infections. However, positive mNGS results frequently present a clinical dilemma because Aspergillus may represent invasive pulmonary aspergillosis (IPA), airway colonization, or transient fungal detection. The clinical value of quantitative fungal burden assessment remains insufficiently defined, particularly regarding risk stratification among untreated patients.

METHODS

We conducted a retrospective real-world cohort study including 114 hospitalized patients with BALF mNGS-positive Aspergillus detected between April 2024 and November 2025. Patients were classified according to clinical IPA diagnosis, antifungal treatment status, and occurrence of respiratory deterioration during a 3-month follow-up period. Quantitative fungal burden was expressed as reads per ten million (RPTM). Receiver operating characteristic (ROC) analysis, logistic regression, integrated discrimination improvement (IDI), and category-free net reclassification improvement (NRI) were used to evaluate diagnostic and prognostic performance.

RESULTS

Among 114 patients, 31 met clinical diagnostic criteria for IPA and 83 were classified as non-IPA. Aspergillus burden was significantly higher in IPA patients than in non-IPA patients (logarithmic scale median RPTM 2.46 vs. 0.30, P < 0.001). ROC analysis identified an exploratory cohort-derived diagnostic threshold of 75 RPTM for IPA discrimination (AUC = 0.853, 95% CI 0.745-0.960). Among 77 patients who did not receive antifungal therapy, 31 experienced respiratory deterioration during follow-up. Higher RPTM values were independently associated with deterioration (adjusted OR = 5.27, 95% CI 1.78-17.06, P = 0.001). An exploratory RPTM threshold of 2.5 showed modest discriminatory ability for subsequent respiratory deterioration, with an AUC of 0.682. Incorporation of quantitative fungal burden significantly improved discrimination and reclassification performance beyond conventional clinical variables. In contrast, baseline RPTM showed no significant association with respiratory deterioration among patients receiving antifungal therapy.

CONCLUSIONS

Quantitative interpretation of Aspergillus-positive BALF mNGS results may provide additional information beyond simple pathogen detection. Two exploratory cohort-derived thresholds were identified: a higher threshold associated with clinical IPA adjudication and a lower threshold associated with subsequent respiratory deterioration among untreated patients. These findings are hypothesis-generating and require external validation before clinical application. RPTM should be interpreted as an adjunctive marker within the overall clinical context rather than as a standalone diagnostic or prognostic threshold.