Microbial cell-free DNA sequencing of bronchoalveolar lavage fluid improves diagnostic yield and may add clinical utility in immunocompromised patients with severe pneumonia

  1. Vijeeth Guggilla
  2. Chiagozie O Pickens
  3. Helen K Donnelly
  4. Anna Pawlowski
  5. Erin Korth
  6. Francisco Martinez
  7. Rebecca K Clepp
  8. GR Scott Budinger
  9. Alexander V Misharin
  10. Nandita R Nadig
  11. Benjamin D Singer
  12. Catherine A Gao
  13. Theresa L Walunas
  14. Richard G Wunderink
  15. The NU SCRIPT Study Investigators

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Abstract

Background

Despite sophisticated standard of care (SOC) testing (including bronchoalveolar lavage (BAL) fluid culture and multiplex PCR), the etiology of pneumonia in immunocompromised patients is frequently unknown. Microbial cell-free DNA (mcfDNA) sequencing increases diagnostic yield in plasma but remains understudied in BAL fluid.

Methods

This was a single center, retrospective, observational cohort study. Residual BAL fluid collected from immunocompromised, mechanically ventilated patients with suspected pneumonia was sent to Karius® for mcfDNA sequencing. Given the retrospective design, mcfDNA sequencing results were unavailable to clinicians. SOC testing results were compared to Karius® BAL (KT-BAL) test reports that classified identified microorganisms as Category One (always pathogenic), Category Two (usually pathogenic), or Category Three (rarely pathogenic).

Findings

228 BAL fluid samples from 155 patients were analyzed. In pneumonia patients, KT-BAL yielded 18 additional Category One organisms, 138 additional Category Two organisms, and 313 additional Category Three organisms compared to SOC. Organisms missed by SOC and identified by KT-BAL included bacterial pathogens (99 patients), clinically relevant DNA viruses (34 patients), non- Candida fungi (11 patients), and parasites (1 patient). Compared to patients with Category One or Two organisms identified by both SOC and KT-BAL (n = 68), patients with organisms identified by KT-BAL alone (n = 87) had significantly more cumulative intubation days (11 [5, 20] days vs. 15 [8, 32] days, p = 0.035).

Interpretation

KT-BAL increased diagnostic yield for immunocompromised patients with suspected pneumonia with suggestion of prolonged respiratory failure in patients with pathogens missed by SOC.

Article activity feed

  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17694597.

    This retrospective single-center cohort study evaluates the diagnostic value of microbial cell-free DNA (mcfDNA) sequencing of bronchoalveolar lavage (BAL) fluid (Karius test) in immunocompromised, mechanically ventilated patients with suspected pneumonia. The authors compare KT-BAL results with standard-of-care (SOC) diagnostics and analyze clinical outcomes. The study finds that KT-BAL substantially increases the diagnostic yield - identifying additional bacterial, viral, and fungal pathogens missed by SOC and that detection of these missed pathogens is associated with longer intubation duration, suggesting potential clinical significance.

    Strengths

    1. Novel and clinically relevant

    The preprint addresses an important gap in pneumonia among immunocompromised patients, where conventional tests frequently fail to identify causative pathogens.

    1. Methodological rigor

    The preprint uses pre-existing prospectively adjudicated cohort, minimizing bias in pneumonia diagnosis and provides a robust foundation for retrospective molecular analyses.

    1. Comprehensive comparison of KT-BAL and SOC

    It provides specific comparisons by organism category, showing where KT-BAL adds the most value (in fungi, opportunistic pathogens, DNA viruses).

    1. Transparency in report/reproducibility

    The inclusion of STROBE compliance, data provenance (PhysioNet) and clear funding/COI strengthens its transparency.

    Weaknesses

    1. Retrospective single-center design

    The retrospective approach in the preprint may raise concerns in selection bias and possibly limit casual interpretation of outcome.  

    1. Lack of clinical adjudication of KT-BAL findings

    The preprint may benefit from prospective validation with clinical correlation to improve diagnostic utility. It is unclear how many of the additional detections were true infections vs colonization/contamination.

    1. Absence of plasma mcfDNA comparison

    Given Karius's established plasma test, a matched analysis could clarify whether BAL fluid offers additional diagnostic yield over noninvasive plasma testing.

    Some potential suggestions

    • While the Karius categories are described clearly, the preprint could include more examples/rationales for these designations (especially Category 3 detections in non-pneumonia controls).

    • Figures 2-4 effectively illustrate the key findings, but it could have clearer legends, particularly specifying sample sizes per subgroup

    • Detailed organism lists (Supplementary Tables 1-3) could be moved into main text to improve accessibility.

    • Reporting exact p-values/effect sizes for major comparisons could improve interpretability. eg) ICU mortality difference is borderline significant → should be interpreted cautiously.

    Competing interests

    The author declares that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The author declares that they did not use generative AI to come up with new ideas for their review.

  2. Version published to 10.1101/2025.11.05.25339543 on medRxiv