Culture and amplification-free bacterial sepsis diagnosis Sepsis is a life-threatening complication of an infection that requires immediate treatment. The identification of the causative microorganism utilizing processes based on blood culture is a keystone of sepsis diagnosis. However, blood culture takes one to five days to provide results. Here, we propose the development of a cultureand amplification-free assay to detect and identify bacteria in one hour directly on whole blood. The new assaywill enable physicians to provide therapy targeting the causative organism days earlier than the currentstandards. The new assay will be based on Single MOLecule Tethering (SMOLT), a novel sample preparation andamplification-free molecular detection method developed at Scanogen. The work proposed in this applicationwill build on the achievements of an ongoing project aimed at developing an assay to detect fungi in whole blood. The SMOLT-Bacteria assay proposed in this application will identify the species, genus or order of the causativeorganism in approximately 90% of bloodstream infections. The new system will be easy-to-use, fully automated,and capable of rapid detection in whole blood without blood culture. Despite the urgent clinical need, there isonly one FDA approved culture-free molecular assay for sepsis diagnosis. In comparison to this test, the SMOLT-Bacteria assay will be 4 times faster, 6 times less expensive, and thanks to its higher multiplexing capability, itwill detect and identify the causative pathogen in twice the number of patients.In our preliminary results, we developed a multiplex assay capable of detecting bacteria directly in whole blood.We demonstrated that the assay is highly specific and sensitive with a limit of detection (LOD) between 1-10CFU/mL, depending on the species, which is as good as the only FDA-approved test. In this project, we willcomplete the development of the assay protocol by including new probes and optimizing the overall assay (Aim1). We will test the manual protocol and develop a fully integrated disposable cartridge and instrument (Aim 2).Finally, we will evaluate the assay in pilot analytical and clinical validation studies (Aim 3). We will work with amultidisciplinary team that includes experts in assay development and instrument development, including theformer Vice President of Engineering at Becton Dickinson, experts in microbiology, sepsis diagnosis andtreatment from Johns Hopkins University and the University of Pittsburgh, as well as engineers from KeyTechnologies. After completing this project, we will further optimize the system for usability and manufacturabilityand conduct analytical and clinical studies for submission to the Food and Drug Administration (FDA). Ifsuccessful, the new automated assay can become part of the standard procedure for the diagnosis of patientswith sepsis and improve patients' outcomes by dramatically reducing the time to targeted antimicrobial therapy.
Public Health Relevance Statement: Project Narrative We propose the development of a novel sample-to-answer test for rapid detection and identification of bacteria directly in whole blood for the diagnosis of bloodstream infections.
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