156. Direct Detection and Quantification of Bacterial Cell-free DNA in Patients with Infective Endocarditis (IE) Using the Karius Plasma Next Generation Sequencing (NGS) Test

Abstract

BackgroundThe variable clinical presentation of IE requires a diagnostic tool that accurately detects a wide range of organisms, including in culture-negative (CN) scenarios. A sensitive molecular diagnostic assay that quantitates pathogen DNA could be a useful tool to diagnose IE and evaluate response to antimicrobial therapy.MethodsWe prospectively enrolled 30 hospitalized adult patients evaluated for acute IE classified using the Duke Criteria. Residual plasma samples within 24 hours and/or fresh whole blood within 48–72 hours of enrollment blood culture were collected. Additional samples were collected every 2–3 days for up to 7 time points until discharge. Samples were shipped to the Karius laboratory (Redwood City, California) for testing. Cell-free DNA was extracted and NGS was performed. Human sequences were removed and remaining sequences were aligned to a curated pathogen database of over 1,000 organisms. Organisms present above a predefined statistical threshold were reported. Quantity of DNA for each reported pathogen was expressed as molecules per microliter.ResultsOf 29 patients eligible for analysis, 18 had prosthetic valves and 7 had implanted cardiac devices. Twenty-four patients had Definite IE. Twenty patients had positive blood cultures (including S. aureus, S. epidermidis, E. faecalis, S. agalactiae, Pantoea ananatis, S. sanguinis, C. albicans); NGS identified the same organism isolated in all 20 patients as well as E. cloacae complex, and E. faecalis in 2 of 4 CN Definite IE patients. For 1 CN patient with Possible IE, NGS identified E. coli. NGS and BC were negative for 4 patients with Rejected IE. NGS identified the IE etiology in patients pretreated with antibiotics up to 20 days prior to sample collection. Pathogen DNA signal was often observed in both initial and repeat plasma samples, while repeat blood cultures remained negative.ConclusionThis novel, cell-free pathogen quantitative NGS plasma assay accurately identified causative organisms in patients with IE, even when blood cultures were negative due to pretreatment with antibiotics. Pathogen DNA, detected in plasma longer than blood culture, is a promising biomarker to aid in the diagnosis and monitoring of IE, particularly culture-negative IE.Disclosures P. Shah, Karius Inc.: Collaborator and Research Contractor, Salary. F. Ruffin, Karius Inc.: Collaborator and Research Contractor, Salary. H. Seng, Karius Inc.: Employee, Salary. D. Hollemon, Karius Inc.: Employee, Salary. L. Winn, Karius Inc.: Collaborator and Research Contractor, Salary. C. Drennan, Karius Inc.: Collaborator and Research Contractor, Salary. K. L. Chan, Karius Inc.: Employee, Salary. H. Quach, Karius Inc.: Employee, Salary. T. Blauwkamp, Karius Inc.: Employee, Salary. G. Meshulam-Simon, Karius Inc.: Employee, Salary. D. Hong, Karius Inc.: Employee, Salary. V. G. Fowler Jr., Merck, Cerexa/Actavis, Pfizer, Advanced Liquid Logis, NIH, MedImmune, Basilea, Karius, Contrafect, Regneron, Genentech, Affinergy, Locus, Medical Surface, Inc., Achaogen, Astellas, Arsa...

Next-generation sequencing of microbial cell-free DNA for rapid noninvasive diagnosis of infectious diseases in immunocompromised hosts

“…Recent reports indicate that NGS measuring microbial cfDNA is useful in the diagnosis of cases of Streptococcus pneumoniae-related hemolytic uremic syndrome, Coxiella burnetii endocarditis, invasive Mycobacterium chimaera infection, Nocardia cyriacigeorgica pneumonia, Capnocytophaga canimorsus sepsis, M. tuberculosis complex and M. haemophilum infections, M. bovis aortitis; Candida spp., Aspergillus spp., non-Aspergillus molds invasive infections; Pneumocystis jirovecii pneumonia (PJP), Toxoplasma gondii infection and chorioamnionitis, among others 24,[26][27][28][29][30][31][32][33] . Among 21 patients with culture-positive infective endocarditis, cfDNA NGS identified the same organism as blood cultures in 20 patients (95% sensitivity) and additionally identified Enterococcus faecalis in one out of the three patients with definitive culture-negative endocarditis 34 . Of note, in this study the cfDNA NGS test identified pathogens causing endocarditis in patients pre-treated with antibiotics up to 30 days prior to initial sample collection.…”

Case Report: Comparison of Plasma Metagenomics to Bacterial PCR in a Case of Prosthetic Valve Endocarditis

“…It is unclear whether clearance of microbial cfDNA could account for the negative mNGS assay. Little is known about the pharmacodynamics of microbial cfDNA, although reports have detected microbial cfDNA for at least 2 weeks and as long as 36 days after treatment of endocarditis ( 29 , 30 ). Inferring clearance in this patient's case is complicated because when she was treated for pneumonia, she did not have evidence of endocarditis; rather, she later presented with increased pulmonary valve obstruction and a new vegetation subsequent to treatment, suggesting progression of the infection despite initial therapy with unknown effects on microbial cfDNA concentration.…”

Next-generation sequencing of microbial cell-free DNA for rapid noninvasive diagnosis of infectious diseases in immunocompromised hosts

“…Recent reports indicate that NGS measuring microbial cfDNA is useful in the diagnosis of cases of Streptococcus pneumoniaerelated hemolytic uremic syndrome, Coxiella burnetii endocarditis, invasive Mycobacterium chimaera infection, Nocardia cyriacigeorgica pneumonia, Capnocytophaga canimorsus sepsis, M. tuberculosis complex and M. haemophilum infections, M. bovis aortitis; Candida spp., Aspergillus spp., non-Aspergillus molds invasive infections; Pneumocystis jirovecii pneumonia (PJP), Toxoplasma gondii infection and chorioamnionitis, among others 24, [26][27][28][29][30][31][32][33] . Among 21 patients with culture-positive infective endocarditis, cfDNA NGS identified the same organism as blood cultures in 20 patients (95% sensitivity) and additionally identified Enterococcus faecalis in one out of the three patients with definitive culture-negative endocarditis 34 . Of note, in this study the cfDNA NGS test identified pathogens causing endocarditis in patients pre-treated with antibiotics up to 30 days prior to initial sample collection.…”