Bronchoalveolar Lavage Metagenomic Sequencing in the Early Post-Lung Transplantation Period: A Pilot Comparison with Microbiologic Culture

BACKGROUND: Conventional bronchoalveolar lavage (BAL) culture has limited sensitivity in lung transplant recipients due to universal antimicrobial prophylaxis and inability to detect non-culturable organisms. Metagenomic sequencing of BAL offers culture-independent pathogen identification, but concordance with conventional culture and clinical utility in the immediate post-transplant period have not been evaluated. METHODS: We prospectively enrolled 19 adult lung transplant recipients undergoing serial bronchoscopies during the immediate post-transplant period. BAL samples (n=33) were tested in parallel with conventional culture, microbial cell-free DNA (mcfDNA) sequencing (Karius Focus BAL; with quantitation determined by research analysis), and Oxford Nanopore sequencing using a unified three-tier organism classification framework. BAL host-response biomarkers were profiled concurrently. RESULTS: Karius reported Tier 1 pathogens in 19/33 episodes (58%) versus 8/33 by conventional culture (24%); organism-level concordance was low (Cohen's kappa 0.18). In 16 episodes where Karius reported a pathogen not recovered by culture, 8 across 3 participants represented predictive reports, defined as the same organism subsequently confirmed as causing invasive infection 2-46 days later, including fatal Pseudomonas pneumonia, Enterococcal surgical site infection, and Candidemia. Nanopore sequencing had low yield due to contaminating human DNA. Quantitative mcfDNA burden correlated positively with multiple alveolar inflammatory biomarkers including total protein, IL-6, and sST2. INTERPRETATION: BAL mcfDNA metagenomics identified clinically relevant pathogens not recovered by conventional culture, including predictive reports and non-culturable organisms. Quantitative mcfDNA signal correlated with alveolar inflammatory mediators, supporting the biological relevance of mcfDNA detection beyond conventional diagnostic classification and a role for metagenomic surveillance in the early post-transplant period.