Plasma Microbial Cell-Free DNA Sequencing from over 15,000 Patients Identified a Broad Spectrum of Pathogens.

Microbial cell-free DNA (mcfDNA) sequencing is an emerging infectious disease diagnostic tool which enables unbiased pathogen detection and quantification from plasma. The Karius Test, a commercial mcfDNA sequencing assay developed by and available since 2017 from Karius, Inc. (Redwood City, CA), detects and quantifies mcfDNA as molecules/µL in plasma. The commercial sample data and results for all tests conducted from April 2018 through mid-September 2021 were evaluated for laboratory quality metrics, reported pathogens, and data from test requisition forms. A total of 18,690 reports were generated from 15,165 patients in a hospital setting among 39 states and the District of Columbia. The median time from sample receipt to reported result was 26 h (interquartile range [IQR] 25 to 28), and 96% of samples had valid test results. Almost two-thirds (65%) of patients were adults, and 29% at the time of diagnostic testing had ICD-10 codes representing a diverse array of clinical scenarios. There were 10,752 (58%) reports that yielded at least one taxon for a total of 22,792 detections spanning 701 unique microbial taxa. The 50 most common taxa detected included 36 bacteria, 9 viruses, and 5 fungi. Opportunistic fungi (374 Aspergillus spp., 258 Pneumocystis jirovecii, 196 Mucorales, and 33 dematiaceous fungi) comprised 861 (4%) of all detections. Additional diagnostically challenging pathogens (247 zoonotic and vector-borne pathogens, 144 Mycobacterium spp., 80 Legionella spp., 78 systemic dimorphic fungi, 69 Nocardia spp., and 57 protozoan parasites) comprised 675 (3%) of all detections. This is the largest reported cohort of patients tested using plasma mcfDNA sequencing and represents the first report of a clinical grade metagenomic test performed at scale. Data reveal new insights into the breadth and complexity of potential pathogens identified.

Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease.

Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.