PROJECT SUMMARY Sepsis is a life-threatening condition caused by bacterial infection of the bloodstream that threatens nearly two million lives in the U.S. annually. Risk of death escalates quickly once bacteria or fungi enter the bloodstream; thus, quick and accurate detection of bloodborne pathogens is critical to inform immediate tailored treatment. However, the current standard practice to detect bloodborne bacteria and fungi requires in vitro culture to measure microbial growth, a process which takes 12-120 hours, with times to a confirmed negative sometimes taking as long as 5 days. Positive cultures then undergo additional tests to determine bacterial identity and antimicrobial susceptibility. This protracted process, which includes significant pre-analytical delays caused by sample transport to off-site microbiology labs and long culture times, represents a critical bottleneck in the diagnostic laboratory workflow, creating significant economic and labor burden to the healthcare system and prolonging the time that patients may be receiving inappropriate treatment (broad spectrum antimicrobial drugs instead of targeted therapy). Aincobio is developing a rapid, near patient in vitro diagnostic test, the BactoPhore, that can be deployed outside of the centralized lab to isolate and detect intact and viable low-concentration bacteria and fungi in blood specimens in 2 hours. BactoPhore achieves enhanced analytical sensitivity by rapidly concentrating pathogens for detection to deliver a positive result quickly. To do this, BactoPhore incorporates a “lab-on-a-chip” system that can isolate microbes from 0.5 mL whole blood using dielectrophoresis (DEP) and detect metabolic signatures using electrochemical impedance spectroscopy (EIS) within a low-cost consumable. A diagnostic prototype of the BactoPhore isolates >90% of target bacteria in 30 minutes and can detect 100 CFUs of E. coli within 2 hours. Further, target microbes pre-concentrated from 10 mL of whole blood can be trapped by DEP in <30 minutes, demonstrating the potential for processing clinically relevant volumes. In this Phase I SBIR, Aincobio will demonstrate feasibility of commercializing the BactoPhore test for label-free, antibody-independent, and culture-independent detection, concentration, and isolation of low abundance and viable bacterial and fungal pathogens by 1) evaluating the integrated BactoPhore prototype with common pathogens spiked into whole blood, and 2) demonstrating that BactoPhore enables rapid and sensitive downstream sequencing of the target microbe by depleting host DNA prior to extraction and library preparation. Completion of these aims will establish proof-of-concept that BactoPhore has analytical sensitivity to confirm infection from whole blood within 2 hours and significantly improve time-to-results for the entire diagnostic workflow from positive result to pathogen identification.