A-269 A sepsis diagnostic platform using host protease activity sensors

Abstract Background Sepsis is a serious condition and a leading cause of death in hospitals worldwide. According to the Centers for Diseases Control and Prevention, at least 1.7 million adults in the United States develop sepsis every year and at least 350,000 adult patients die from sepsis. Early and accurate diagnosis is crucial for timely intervention and improved patient outcomes. Current diagnostic approaches rely on clinical criteria and conventional biomarkers such as procalcitonin (PCT), lactate, and C-reactive protein (CRP), which lack the specificity needed to differentiate sepsis from non-infectious system inflammatory response syndrome (SIRS). Additionally, the gold standard for pathogen identification, blood culture-based diagnostics, are often time-consuming and yield negative results in a significant proportion of cases. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has shown promising results in microbial identification and biomarker discovery for infectious diseases with optimal analytical sensitivity and specificity. Our study aims to develop a novel MALDI-TOF MS based assay targeting human proteases, key enzymes involved in the inflammatory and proteolytic processes of sepsis. By quantifying the activity of theses enzymes in plasma, this technique provides a faster, more sensitive, specific and cost-effective diagnostic approach for sepsis diagnosis. Methods 40 sepsis patients and 40 non-sepsis control patients’ blood were collected in lithium heparin plasma tube and stored at 4 °C until analysis. The samples were incubated with customized HNE, MMP, and cathepsin sensors, which are molecular probes designed to undergo cleavage in the presence of their respective target enzymes. The resulting products were analyzed using MALDI-TOF MS to detect and quantify the enzyme activities. The assay sensitivity, specificity, and receiver operating characteristic (ROC) curve analysis were performed. Results MALDI-TOF-MS based detection of HNE and MMP activity demonstrated superior diagnostic performance compared to lactate assays. The combined analysis of these two enzymes significantly improved the differentiation between sepsis and non-sepsis patients, achieving higher sensitivity and specificity compared to lactate assay. The area under the ROC curve (AUC) showing >90%, indicating great diagnostic potential of this platform for our assay. Conclusion This study demonstrates out MALDI-TOF MS-based assay offers a superior diagnostic tool for sepsis. The method provides higher sensitivity and specificity. Additionally, our assay is more cost-effective, not to mention the potential to further cutting cost associated with prolonged hospital stays and unnecessary antibiotic use due to misdiagnosis. Our assay offers the rapid, precise, and affordable sepsis detection, showing the potential to improve sepsis patient outcomes.