Highly Multiplexed Single Molecule Tethering

Highly Multiplexed Single Molecule Tethering Summary Bloodstream infections in patients with sepsis symptoms should be promptly treated with antibiotics; however, the time required to identify pathogens using methods based on blood culture is a major obstacle to timely effective antimicrobial therapy. The development of a comprehensive molecular assay capable of identifying microbes directly from whole blood has been elusive despite major efforts due to the low concentration of microbes in the bloodstream and the large number of microbial species that cause bloodstream infection (200+). We have developed an automated assay that detects 13 bloodstream microbes directly in whole blood at the low concentrations found in clinical samples, with LODs between 0.5 and 3 CFU/mL and a turnaround time of 70 minutes. The assay performance is superior to other FDA cleared assays for bloodstream pathogen detection. The new assay is based on Single MOLecule Tethering (SMOLT), a novel sample preparation and amplification-free molecular detection technology developed at Scanogen. Here, we propose to dramatically increase the multiplexing capacity of SMOLT using a novel approach with the goal of producing a panel that covers the organisms responsible for more than 99% of bloodstream infection cases (Aim 1). We will also integrate the automated assay into a single disposable cartridge that will be processed by an easy-to-use and fully automated desktop instrument (Aim 2). We will evaluate the new assay in pilot clinical studies where we will directly compare the performance of the new assay to the standard of care diagnostic test based on blood culture followed by mass spectroscopy (Aim 3). Each of these specific aims has quantitative milestones. Our long-term goal is the development of an FDA-cleared and CLIA-waived system; this project is a critical step towards that goal. We will address the challenges of achieving the required level multiplexing and assay integration, which are the most serious risks on the path to developing the clinical system. We will work with a multidisciplinary team that includes experts in assay development and instrument development, including the former Vice President of Engineering at Becton Dickinson, experts in microbiology, sepsis diagnosis and treatment from Johns Hopkins University and the University of Pittsburgh, as well as engineers from Key Technologies. After completing this project, we will further optimize the system for usability and manufacturability and conduct analytical and clinical studies for submission to the FDA. If successful, the new automated assay can become part of the standard procedure for the diagnosis of patients suspected of having sepsis. The new highly multiplexed assay will be useful in other applications where rapid, cost-effective, and highly multiplexed molecular detection is needed.