A-108 Comprehensive Performance Evaluation of a Rapid, Universal, Sepsis Diagnostic Workflow that identifies Pathogens and characterizes their Antimicrobial Resistance

Abstract

Rapid and accurate diagnosis of bloodstream infections (BSIs) is essential to initiate appropriate therapy in patients with sepsis, where mortality risk increases with every passing hour. The current gold standard, blood culture, requires approximately 15–72 hours for pathogen identification (ID), followed by an additional 8–15 hours for antimicrobial susceptibility testing (AST). In addition to its prolonged turnaround time (TAT), blood culture is susceptible to both false-positive and false-negative results. Furthermore, most nucleic acid–based assays available commercially rely on blood culture as the initial input material and/or are restricted to detecting a predefined panel of pathogens and antimicrobial resistance (AMR) genes. Therefore, a rapid, unbiased diagnostic approach capable of directly detecting pathogens and their associated AMR markers from whole blood within a clinically actionable TAT remains a significant unmet need in sepsis management. 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 that can report pathogen ID and AMR determinants, directly from whole blood, within 6 to 8 hours using nanopore sequencing. The workflow is illustrated in Figure-1. We evaluated analytical sensitivity 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 (58 Blood cultures and 31 Whole blood samples) to evaluate the clinical concordance of the ASPIRE workflow with blood culture outcomes. In addition, ASPIRE workflow was tested with 31 clinical bacterial isolates which were pre-characterized for their antimicrobial signatures (19 confirmed positives and 12 confirmed negatives). A breadth of coverage (BoC) analysis for this workflow was also performed using 19 commercially procured bacterial isolates. AMR detection: The ASPIRE system displayed the ability to simultaneously identify multiple relevant AMR determinants in a panel of blinded multi drug resistant bacterial isolates with a high degree of confidence. Except for the specific variant IMP-13, all targeted markers identified in the truth key were correctly identified Analytical sensitivity: The ASPIRE system achieved detection of 5 to 10 CFU/mL for ESKAPE bacteria and Candida albicans using the ASPIRE workflow. Clinical sensitivity: Preliminary clinical studies showed substantial concordance between blood cultures and ASPIRE workflow at 87.8% (58 samples), sensitivity of 94% (47/50 confirmed positives) and specificity of 50% (4/8 confirmed negatives) when tested with 58 blood culture samples. We observe a PPA of 0.94 and NPA of 0.8. With ∼1mL of direct (uncultured) blood, we observed concordance of 83.9% (31 samples), sensitivity of 73.7% (14/19 confirmed positives) and specificity of 100% (12/12 confirmed negatives). Breadth of Coverage: The ASPIRE workflow showed 100% detection across 19 commercially available bacteria. ASPIRE is a novel, cutting-edge, rapid, and universal Sepsis diagnostic workflow that can identify pathogens and characterize their AMR, directly from whole blood. The TAT of 6 hours positions ASPIRE workflow as a unique solution that can enable timely clinical interventions through sensitive detection of broad range of pathogens.