Microbial Cell-Free DNA Sequencing for Diagnosing Lung Infections in Immunocompromised Patients

Rationale Rapid and comprehensive detection of respiratory pathogens is essential for immunocompromised patients with suspected pneumonia. Conventional diagnostic methods are limited in scope, often slow, and lack sensitivity, leading to reduced diagnostic yield.

To analytically and clinically validate a microbial cell-free DNA (mcfDNA) sequencing assay for bronchoalveolar lavage fluid (BALF) that enables broad and sensitive detection of pathogens causing pneumonia in immunocompromised patients.

The BALF-mcfDNA assay detects more than 500 bacteria, DNA viruses, fungi, and parasites. Analytical validation used 324 contrived samples containing DNA from 11 representative organisms and 1,294 in-silico samples. Clinical validation compared BALF-mcfDNA sequencing with usual care (UC) testing in 118 immunocompromised adults with suspected pneumonia. An independent expert panel adjudicated the etiologic diagnoses. Measurements and Main Results BALF-mcfDNA testing demonstrated high inclusivity (97%), exclusivity (96%), and precision (coefficient of variation <15%), with linear quantification across four orders of magnitude. Limits of detection were consistent across taxa, GC content, and genome size. In clinical evaluation, BALF-mcfDNA sequencing identified probable pneumonia pathogens in 34% of participants versus 24% for UC testing, increasing diagnostic yield by 43% (57% when combined with UC). In 24% of concordant cases, mcfDNA sequencing provided additional clinically relevant information by identifying new or species-level pathogens.

BALF-mcfDNA sequencing enables sensitive, comprehensive, and rapid pathogen detection from BALF specimens, outperforming conventional methods and improving etiologic diagnosis of pneumonia in immunocompromised hosts. Competing Interest Statement KB, KHJ, FN, IDV, MLV, AZ, VP, BAP, SB and TB are employees of and shareholders in Karius. JDG and JAH served as members of the adjudication panel for this manuscript. JAH and SB report research grants paid to their institution from Karius. JAH reports consulting for Karius and support for travel to advisory board meetings from Karius. Funding Statement This work was supported by Karius Inc, Redwood City, California, USA. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All participants in the PICKUP study provided written informed consent. Ethical review and oversight were conducted by Advarra. The ethical committees of all participating institutions gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors.