Shotgun metagenomic sequencing analysis as a diagnostic strategy for patients with lower respiratory tract infections

Introduction: Diagnosing lower respiratory infections (LRIs) using conventional diagnostic methods (CDMs) presents limitations in detecting suspected pathogens. This study compares the latest CDMs with shotgun metagenomic sequencing (SMS) for bronchoalveolar lavage (BAL) fluid. The primary objective is to enhance pathogen detection using SMS. Materials and Methods: A total of 16 BAL fluid samples from patients with pneumonia with positive results in various CDMs—bacterial/fungal cultures, real-time PCR for Mycobacterium tuberculosis, cytomegalovirus, or the BioFire® FilmArray Pneumonia Panel—were included. Samples were subjected to 10 Gb SMS on the NovaSeq 6000 (Illumina) and were aligned against the NCBI RefSeq database. For eukaryotic reads, an additional matching process was performed using the internal transcribed spacer (ITS) region of fungi. Antibiotic resistance genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database model. To identify significant pathogens, thresholds for the relative abundance of SMS reads were applied to evaluate the concordance between CDM- and SMS-detected microbes. Results: The proportion of microbial reads ranged from 0.00002–0.04971% per sample. SMS detected corresponding bacterial reads (2–23,869) with relative abundance between 0.02% and 87.5%. Eukaryotic reads varied from 0 to 32, with no fungal alignment at the genus level. Candida species were identified in four samples using ITS. No viral reads were detected. In 10 out of 16 cases (63%), SMS detected pathogens above the threshold by SMS. When subdominant taxa were included, SMS detected pathogens in 11 out of 16 cases (69%). ARGs meeting perfect criteria via the Resistance Gene Identifier were observed in two cases. Conclusion: This study represents the first comparison of SMS and CDMs, including the FilmArray Pneumonia Panel, in the context of LRI diagnostics. SMS may serve as a valuable supplementary tool for LRI diagnosis. Further research is necessary to improve sensitivity and cost-effectiveness.