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

Abstract

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.

Keywords: high-throughput nucleic acid sequencing; liquid biopsy for infectious diseases; metagenomics; microbial cell-free DNA.

FIG 1

Number of Karius Tests ordered by state from April 2018 to September 2021. No samples were received from the states shaded in blue.

FIG 2

Number of detections by the Karius Test of the different supergroups of taxa from April 2018 to September 2021. n = 22,792: bacteria, 16,221; viruses, 4,737; fungi, 1,758; and parasites, 70. Percentages reflect the proportion of total number of detections spanning 701 microbial taxa.

FIG 3

Number of detected taxa counts per report for all positive reports, April 2018 to September 2021. Percentages reflect the proportion of all positive reports (n = 10,752).

FIG 4

Frequency distribution of Legionella-like organisms detected, n = 80 (<1% of all bacterial detections, n = 16,203), April 2018 to September 2021. *indicates species not associated with human infections.

FIG 5

Frequency distribution of Nocardia spp. detections, n = 76 (<1% bacterial detections, n = 16,203), April 2018 to September 2021. An asterisk (*) indicates species more frequently isolated from clinical specimens (69). A dagger (†) indicates species-specific susceptibility patterns (51).

FIG 6

Frequency distribution of Mycobacterium spp. detections, n = 156 (1% of all bacterial detections, n = 16,203), April 2018 to September 2021. Asterisk (*) indicates slowly growing mycobacteria of established clinical significance (70). Dagger (†) indicates rapidly growing mycobacteria considered common human pathogens. Double dagger (‡) indicates rapidly growing mycobacteria considered less common or rare human pathogens (71).

FIG 7

Frequency distribution of zoonotic and vector-borne bacteria detections, n = 247 (2% of all bacterial detections, n = 16,203), April 2018 to September 2021. Asterisk (*) indicates bacteria causing a nationally notifiable infectious disease (72).

FIG 8

Frequency distribution of Candida spp. detections, n = 648 (36% of all fungal detections, n = 1,776), April 2018 to September 2021.

FIG 9

Frequency distribution of Aspergillus spp. detections, n = 374 (21% of all fungal detections, n = 1,776), April 2018 to September 2021. Asterisk (*) indicates most common pathogenic species (73).

FIG 10

Frequency distribution of detections in the order Mucorales, n = 196 (11% of all fungal detections, n = 1,776), April 2018 to September 2021. Asterisk (*) indicates taxa implicated in human mucormycosis (74).

FIG 11

Frequency distribution of detections of systemic dimorphic fungi, n = 78 (4% of all fungal detections, n = 1,776), April 2018 to September 2021.

FIG 12

Frequency distribution of detections of dematiaceous fungi, n = 33 (2% of all fungal detections, n = 1,776), April 2018 to September 2021.

FIG 13

Frequency distribution of detections of protozoan parasites, n = 57 (89% of all eukaryotic parasite detections, n = 64), April 2018 to September 2021.