The Utility of Real-time PCR, Metagenomic Next-generation Sequencing, and Culture in Bronchoalveolar Lavage Fluid for Diagnosis of Pulmonary Aspergillosis

Abstract

Timely detection of Aspergillus infection is crucial given the high mortality rate of pulmonary aspergillosis (PA). Here, the diagnostic performances for PA of mycological culture, Aspergillus real-time Polymerase Chain Reaction (RT-PCR), and metagenomic next-generation sequencing (mNGS) assay from bronchoalveolar lavage fluid (BALF), were evaluated. Totally 139 patients with suspected fungal pneumonia were enrolled between December 2021 and July 2023, collecting 139 BALF samples for RT-PCR and culture, with 87 undergoing mNGS assay. The sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve (AUC) with 95% confidence intervals of these assays for PA were as follows: 35.3% (14.2–61.7%), 100.0% (94.0–100.0%), 100.0% (54.1–100.0%), 84.5% (79.3–88.6%), and 0.676 (0.560–0.779) for culture; 82.4% (56.6–96.2%), 98.3% (91.1–100.0%), 93.3% (66.4–99.0%), 95.2% (87.6–98.2%) and 0.903 (0.815–0.959) for same diagnostic performance of RT-PCR and mNGS; and 94.1% (71.3–99.9%), 96.7% (88.5–99.6%), 88.9% (67.1–96.9%), 98.3% (89.6–99.7%), 0.954 (0.880–0.989) for RT-PCR combining mNGS; RT-PCR, mNGS, and their combination significantly improved in AUC values over culture (p <0.001), but RT-PCR testing and mNGS had no significant difference with each other and their combination. Overall, the performance of culture was limited by low sensitivity, both RT-PCR and mNGS assays as single diagnostic tests are promising compared to culture and combined tests.