Integrated genotype-informed single-cell molecular antibacterial susceptibility testing platform for rapid and actionable diagnosis of bacteremia - Project Summary Bacteremia, the presence of living bacteria in blood, can lead to life-threatening sepsis and represents a primary cause of death despite advances in modern medicine. For the diagnosis of bacteremia, the current gold standard is blood culture. Unfortunately, blood culture is insensitive and can take up to 5 days to complete. During this lengthy time window, patients may continue receiving ineffective or unnecessary broad-spectrum antimicrobials, leading to poor treatment outcomes, adverse iatrogenic effects, and increased selection for multi-drug-resistant “superbugs”. Molecular diagnostics promise to speed up the diagnosis and improve the treatment of bacteremia. To date, however, such a promise remains unfulfilled because existing molecular diagnostics cannot simultaneously accomplish: 1) rapid identification of species of the bacteria from a broad panel of potential causative bacteria, 2) prompt testing of the susceptibilities of the identified bacteria against various antibacterial agents, and 3) “needs-driven” versatility to adjust test specifications accordant with clinical context. A better molecular diagnostic platform is therefore urgently needed for the acute management of bacteremia. In response, we propose to develop an integrated molecular and single-cell detection platform capable of rapid bacterial detection, species identification (ID), bacterial load quantification, and antibacterial susceptibility testing (AST) in a streamlined test that allows customizable workflow. First, the proposed platform will perform bacteria ID and measure bacterial load from blood via ultrafast magnetofluidic PCR and “probe melt analysis” (PMA) within a magnetofluidic ID device in 30 min. This device is composed of a low-cost cartridge and an automated instrument. The cartridge and the instrument are designed to perform bacteria lysis, magnetic-based DNA extraction and purification, and PCR-PMA – a unique strategy that detects each bacterial species by a DNA probe with a specific melting temperature and fluorescence color code. The species ID and bacterial load provides guidance and specifications for downstream AST. Next, the proposed platform will conduct “single-cell molecular AST” to quantitatively detect bacterial ribosomal RNA as a surrogate viability marker at the single cell level, which has the potential to accelerate testing time to below the time scale of bacteria replication. Single-cell molecular AST will be performed within a microfluidic chip and a companion instrument that can automate the assay steps – including antibacterial exposure, bacteria lysis, and single-cell RT-PCR-PMA – in 90 – 150 min. The proposed molecular detection platform can provide timely and objective data to clinicians during acute care of bacteremia, which can improve their ability to establish diagnosis and administer antibacterial treatments, potentially improving clinical outcomes and curtailing the emergence of multi-drug resistant bacteria.
