Can Next-Generation Sequencing (NGS) be performed on a sample obtained from a Bronchoalveolar Lavage (BAL)?

Next-Generation Sequencing on Bronchoalveolar Lavage Samples

Yes, Next-Generation Sequencing (NGS) can be performed on bronchoalveolar lavage (BAL) fluid and represents an emerging diagnostic tool for comprehensive pathogen detection in respiratory infections, particularly when conventional testing fails to identify causative organisms. 1

Technical Feasibility and Applications

NGS technology has been successfully applied to BAL samples for multiple clinical applications:

• Metagenomic NGS (mNGS) enables pathogen-agnostic detection directly from BAL fluid, identifying bacteria, fungi, viruses, and mixed infections that conventional culture methods frequently miss 2, 3
• BAL fluid is considered a sterile specimen type that is particularly suitable for NGS-based pathogen detection in respiratory infections 3
• The technology can identify all pathogens simultaneously based on sequence homology, potentially replacing multiple conventional laboratory tests 1

Clinical Performance in BAL Samples

The diagnostic performance of NGS on BAL fluid demonstrates significant advantages over conventional testing:

• For bacterial detection in ventilator-associated pneumonia, mNGS showed 97.1% sensitivity (95% CI: 93.2-101.0%) compared to conventional testing, with a negative predictive value of 94.1% 2
• mNGS detected pathogens in 58% of BAL samples where conventional culture methods were negative, particularly important for patients already receiving empiric antimicrobial therapy 2
• Viral pathogens and mixed infections are readily identified through NGS, with studies detecting viruses in 30-42% of cases that would be missed by standard bacterial culture 2, 4

Specific Clinical Scenarios

NGS on BAL fluid has proven particularly valuable in challenging diagnostic situations:

• For immunocompromised patients with pulmonary infections, including those with hematological malignancies or dermatomyositis, mNGS detected multiple fungal pathogens (Pneumocystis jirovecii, Aspergillus fumigatus, Rhizopus oryzae) when conventional testing failed 1, 5
• In pediatric respiratory failure cases, NGS identified complete viral genomes (enterovirus D68) and detected candidate pathogens in patients where conventional methods were non-diagnostic 3
• Detection rates in BAL samples reached up to 87% for respiratory specimens when using mNGS technology 1

Quality Assurance Considerations

Implementation of NGS on BAL samples requires attention to quality standards:

• Complexities of NGS assays require evolving standards to ensure testing quality, including validation procedures, quality control, and proficiency testing 1
• Turnaround time for commercial plasma cell-free DNA testing is rapid (days), though operational requirements may extend overall time to weeks, similar to conventional diagnostics 1
• Environmental controls and stringent processing protocols are essential, including enrichment for microbial DNA and alignment to curated databases containing ≥1000 microbes 1

Integration with Conventional BAL Testing

NGS should complement, not replace, standard BAL procedures:

• The American Thoracic Society recommends BAL with differential cell count as a foundational diagnostic test for interstitial lung disease, with NGS serving as an adjunctive molecular diagnostic approach 1, 6
• BAL samples should be processed promptly (within 4 hours for suspected fungal infections) regardless of whether conventional or NGS testing is performed 6
• A minimum of 5 mL pooled BAL sample is needed for cellular analysis, with optimal volume of 10-20 mL, which can accommodate both conventional and NGS testing 1

Common Pitfalls and Caveats

Several important limitations must be recognized:

• Correlation between NGS and conventional culture is often poor, with studies showing the same predominant bacterial strain identified by both methods in only a single sample out of 12 tested 7
• False-positive results can occur from cell-free DNA originating from non-tumor sources, including clonal hematopoiesis, leading to detection of KRAS and TP53 mutations that may not represent true infection 1
• NGS results must be interpreted in clinical context, as detection of microbial DNA does not always indicate active infection versus colonization or contamination 2, 5
• Standardization remains incomplete, with variation in gene coverage, analytical pipelines, and interpretation guidelines across different NGS platforms 1