Analytical and Clinical Validation of Direct Detection of Antimicrobial Resistance Markers by Plasma Microbial Cell-free DNA Sequencing

Sequencing of plasma microbial cell-free DNA (mcfDNA) has gained increased acceptance as a valuable adjunct to standard-of-care testing for diagnosis of infections throughout the body. Here we report the analytical and clinical validation of a novel application of mcfDNA sequencing, the non-invasive detection of seven common antimicrobial resistance (AMR) genetic markers in 18 important pathogens with potential to harbor these markers. The AMR markers include SCC mec , mecA and mecC for methicillin, vanA and vanB for vancomycin, bla CTX-M for oxyimino-cephalosporin and aztreonam, and bla KPC for carbapenem resistance. The AMR markers are computationally linked to the pathogens detected, using a statistical model based on observed AMR gene and pathogen abundances. Analytical validation showed high reproducibility (100%), inclusivity (54 to100%), and exclusivity (100%), with limits of detection ranging from 425 to 6,107 pathogen mcfDNA molecules/μL for the different markers. Clinical accuracy was assessed with 115 unique plasma samples from patients at 7 study sites with concordant culture results for 12/18 (66.7%) target bacteria from a variety of specimen types and correlated with available phenotypic antimicrobial susceptibility test results and genotypic results when available. The positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA), and diagnostic yield (DY) were estimated for each AMR marker. The results for the combination of SCC mec and mecA for staphylococci were PPA 19/20 (95.0%), NPA 21/22 (95.4%), OPA 40/42 (95.2%), DY 42/60 (70.0%); vanA for enterococci were PPA 3/3 (100%), NPA 2/2 (100%), OPA5/5 (100%), DY 5/6 (83.3%); bla CTX-M for gram-negative bacilli were PPA 5/6 (83.3%), NPA 29/29 (100%), OPA34/35 (97.1%), DY 35/49 (71.4%); and bla KPC for gram-negative bacilli were PPA 0/2 (0%), NPA: 23/23 (100%), OPA23/25 (92.3%), DY 25/44 (56.8%). The addition of AMR capability to plasma mcfDNA sequencing should provide clinicians with an effective new culture-independent tool for optimization of therapy. ### Competing Interest Statement The following authors declare a conflict of interest: All employees of Karius, which markets the commercial test evaluated in this study, shared responsibilities for study design, analysis of the data, and writing and editing of the manuscript. John Joseph Farrell (OSF Healthcare), Sanjeet Dadwal, (City of Hope), Amy L. Carr, (AdventHealth Orlando), Arryn Craney (Orlando Health), Matthew Pike (Carle Foundation Hospital), James B. Wood (Indiana University School of Medicine) received grant support from Karius to support the collection and curation of orthogonal data that were fundamental to the clinical validation and reviewed the manuscript. This work was supported solely by funding from Karius. ### Funding Statement This study was funded by Karius. ### 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: The study was conducted in compliance with good clinical practice guidelines set forth by the International Conference on Harmonization (ICH-E6). The following Institutional Review Boards granted waiver of consent for the collection of additional clinical data to support this study: AdventHealth Institutional Review Board, City of Hope Institutional Review Board, Orlando Health Institutional Review Board, Peoria Institutional Review Board, and Advarra Institutional Review Board. The Indiana University Institutional Review Board and the Duke Institutional Review Board granted study approval with the ability to use the study samples for additional future research upon consent. In addition, the sample identifiers reported here we unknown to anyone outside of the research group. 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