663. Two (Plus) Birds, One Stone: The Rapid, Comprehensive, Non-invasive Detection of Co-Pathogens of Critical Importance Using A Plasma-based Microbial Cell-free DNA Next-generation Sequencing Test

Abstract Background Immunocompromised (IC) patients are at risk for infections by a spectrum of invasive pathogens. The overlap in presentation makes it challenging to differentiate among infectious etiologies and critical co-infections (CI) may remain undiagnosed. Open-ended, comprehensive assessment of infection through microbial cell-free DNA (mcfDNA) next-generation sequencing (NGS) of plasma offers the potential for the rapid identification of multiple co-infecting pathogens of critical importance (CI-POCI) with one test. Methods Karius TestTM (KT) results from patients who underwent clinical testing from December 2016 to April 2021 were reviewed for detections of two or more CI-POCI including parasites, fungi (Pneumocystis jirovecii, Trichosporon sp, endemic mycoses, Aspergillus sp., Mucorales, Non-Aspergillus/Non-Mucorales molds), mycobacteria, Legionella sp., Nocardia sp. and Listeria. KT, developed and validated in Karius’ CLIA certified/CAP accredited lab, detects mcfDNA from plasma. McfDNA is extracted, NGS performed, human sequences removed and remaining sequences aligned to a curated pathogen database of > 1500 organisms. Organisms present above a statistical threshold are reported and quantified. For > 85% of tests the time to result reporting is the next day from sample receipt. Results KT detected CI of two or more POCI in 59 samples (75% adults, 25% children). The most common partnering co-pathogens of critical importance were Aspergillus sp (38), Mucorales (17) and PJP (14); the most common combinations were two or more distinct Aspergillus sp (14) followed by an Aspergillus sp and a Mucorales (12). There were 17 samples with the detection of three or more CI-POCI (29%). Figure 1. Chord Plot of Co-infections with Pathogens of Critical Importance The outer circle sections represent Karius Test detections belonging to different taxonomic groups. The length of each circle section is proportional to the total number of detections of a taxon belonging to that group. The chords connecting a pair of circle sections are proportional to the number of times two taxa from those groups were observed together, weighted by the total number of taxa detected. Conclusion Plasma mcfDNA NGS offers a rapid, comprehensive non-invasive means of detecting CI-POCI in IC patients with one test. Although rare, co-infections with POCI can greatly increase mortality. The KT can provide important insights into pathogen-pathogen interactions in complex hosts and help optimize therapy. Disclosures Matthew Smollin, PharmD, Karius, Inc. (Employee) Martin S. Lindner, PhD, Karius, Inc. (Consultant) Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Castillo-Galvan, MD MPH, Karius Inc. (Consultant) Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Aparna Arun, MD, Karius (Employee)


Figure 1. Chord Plot of Co-infections with Pathogens of Critical Importance
The outer circle sections represent Karius Test detections belonging to different taxonomic groups. The length of each circle section is proportional to the total number of detections of a taxon belonging to that group. The chords connecting a pair of circle sections are proportional to the number of times two taxa from those groups were observed together, weighted by the total number of taxa detected.
Conclusion. Plasma mcfDNA NGS offers a rapid, comprehensive non-invasive means of detecting CI-POCI in IC patients with one test. Although rare, co-infections with POCI can greatly increase mortality. The KT can provide important insights into pathogen-pathogen interactions in complex hosts and help optimize therapy. Background. Metagenomic next-generation sequencing (mNGS) of plasma cellfree DNA has significant potential to improve infectious diseases diagnostics through unbiased detection of pathogens. However, the optimal patient population or clinical condition for this testing has not been determined.
Methods. We performed a retrospective review of all orders for plasma cell-free DNA mNGS using the Karius test (Karius, Redwood City, CA) from The Children's Hospital of Philadelphia from 7/1/19-4/30/21. Chart review then determined if the test had a positive, negative, or no clinical impact.
Results. 25 mNGS tests were ordered on 24 unique patients. The majority of tests were ordered on immunocompromised patients (Table 1). Most mNGS tests were ordered after completion of routine microbiological testing (17/25, 71%). Three tests were not completed as ordered. Most completed tests (18/22, 82%) had no impact on clinical care as they confirmed the known diagnosis or were not acted upon (Figure 1). mNGS testing had a positive impact in 2 cases. For one patient with congenital heart disease presented with persistent fever and concern for endocarditis despite negative infectious workup, a negative mNGS result allowed for continued monitoring without therapy. Another patient with a lymphatics disorder had mNGS performed due to persistent clinical instability; testing was positive for Candida parapsilosis, allowing for early initiation of antifungal therapy. However, test results had a negative clinical impact in 2 other patients. In a patient with congenital heart disease and fever, identification of two organisms led to prolonged antibiotic therapy for endocarditis without resolution of symptoms. In a patient with leukemia, report of a dematiaceous mold led to further diagnostic testing, including a lumbar puncture, as well as treatment with antifungal therapy despite no clear diagnosis. Table 1 Conclusion. In this study, the majority of plasma cell-free mNGS tests had no impact on clinical care. mNGS testing did positively impact care in 2 patients, but did had a negative impact on care in 2 instances, leading to further testing and unnecessary treatment. Further investigation is needed to determine the ideal population or clinical condition for testing and the ideal time of sending plasma cell-free mNGS tests. Background. Clinical microbiology traditionally relies on culture methodology and serological testing, that have inherent limitations. Newer diagnostic techniques such as Next Generation Sequencing (NGS) have shown promise to improve microbial identification. In select scenarios, we send clinical specimens to reference laboratories for NGS testing in addition to current standard of care (SOC) diagnostics. We wanted to determine how this diagnostic approach has impacted patient care. We also wanted to review the financial burden through cost-benefit analysis for these 'send-out' tests.

Clinical and Financial Impact of Next Generation Sequencing (NGS) in addition to Conventional Microbiology Testing in our Urban Referral Health Center
Methods. We performed a retrospective chart review of all cases over a 3-year period in which NGS was submitted. Data, including demographics, comorbidities, antimicrobial use, and diagnosis (by SOC and NGS) were gathered. We delineated how often there was concordance or discordance between SOC and NGS. We also obtained