A-321 Performance evaluation of a rapid, universal and automation amenable sepsis diagnostic workflow that identifies pathogens and determines their antimicrobial resistance directly from whole blood

Background Fast and accurate diagnosis of bloodstream infections (BSIs) is critical for administering an appropriate treatment regimen in patients, who face hourly-increasing mortality risk due to sepsis. The current gold standard technique is blood culture, and it takes 15 to 72 hours for pathogen identification (ID) and an additional 8 to 15 hours for antimicrobial susceptibility (AST). Apart from the long turnaround time, blood culture is prone to false positives and negatives. Most of the nucleic acid-based methods in the market also require blood culture as a starting sample and/or are limited in identifying pre-defined set of pathogens and antimicrobial resistance (AMR) genes. Hence, the availability of a rapid, agnostic pathogen diagnostic for sepsis which detects pathogens and associated AMR directly from whole blood in a clinically actionable turnaround time, remains a major unmet need in managing critically ill patients. Towards advancing the field of sepsis diagnostics, we have developed a cutting-edge approach called ASPIRE: Agnostic Sepsis Pathogen Identification and REsistance determination. This workflow is rapid, agnostic, quantitative, automation amenable and based on nanopore sequencing that can report pathogen ID and AMR determinants, directly from whole blood, within 6 to 8 hours. The workflow is illustrated in figure 1. Methods We evaluated the performance of ASPIRE workflow using 3 to 5 mL contrived blood samples (n=50) spiked with typical BSI bacteria and fungi at 10 to 100 CFU/mL. We also tested =1.0 mL clinical samples (n=50 confirmed positives and 10 confirmed negatives, tested as blinded samples) to evaluate the clinical concordance of the ASPIRE workflow with blood culture outcomes. Results The ASPIRE workflow can detect 10-100 CFU/mL of spiked bacteria and fungi in 6 to 8 hours turnaround time. We have achieved removal of 95 to 99% human genomic DNA from 3 mL blood samples. Multiple AMR determinants were also detected within blinded clinical samples. We observed significant concordance with clinical samples. Conclusion ASPIRE is a novel, cutting-edge, rapid, and universal Sepsis diagnostic workflow that can identify pathogens and characterize their AMR, directly from whole blood. Clinical studies showed significant concordance between blood cultures and ASPIRE workflows. This work has been supported by NIH-NIAID funding under the contract 75N93023C00025