Culture and amplification-free bacterial sepsis diagnosis

Culture and amplification-free bacterial sepsis diagnosis Summary 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 culture and amplification-free assay to detect and identify bacteria in one hour directly on whole blood. The new assay will enable physicians to provide therapy targeting the causative organism days earlier than the current standards. The new assay will be based on Single MOLecule Tethering (SMOLT), a novel sample preparation and amplification-free molecular detection method developed at Scanogen. The work proposed in this application will 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 causative organism 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 is only 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, it will 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 1-10 CFU/mL, depending on the species, which is as good as the only FDA-approved test. In this project, we will complete the development of the assay protocol by including new probes and optimizing the overall assay (Aim 1). 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 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 Food and Drug Administration (FDA). If successful, the new automated assay can become part of the standard procedure for the diagnosis of patients with sepsis and improve patients’ outcomes by dramatically reducing the time to targeted antimicrobial therapy.