A-107 Development of preanalytics for a Rapid, Integrated Sepsis Diagnostic and Prognostic Workflow combining Pathogen Detection with Host Immune Response Profiling

Abstract

Sepsis is a life-threatening syndrome driven by a dysregulated immune response to infection and remains a major global health burden. WHO estimates indicate that sepsis accounts for ∼20% of global deaths, affecting 49 million people and causing 11 million deaths annually. Early identification of both infecting pathogen and severity of illness is essential in austere settings that include military medicine where symptomatic patients can be prioritized for treatment or transport. The current gold standard, blood culture, requires 0.5 to 5 days for pathogen identification and antimicrobial susceptibility testing, leading to critical delays for decision-making. There is no single commercially available integrated solution that can identify pathogens and assess sepsis severity. To address this gap, we developed INSPIRE (INtegrated Sepsis Pathogen identification and Immune Response Evaluation) workflow, a 6-hour sequencing-based prognostic and diagnostic assay that identifies pathogens, detects antimicrobial resistance (AMR), and predicts patient mortality directly from whole blood. This test is unbiased and designed to identify most sepsis causing pathogens and is compatible with use in low resource settings. INSPIRE workflow (Figure-1) achieves pathogen identification using universally amplified DNA signatures and predicts sepsis severity through relative expression profile of 18 clinically relevant host mRNAs using nanopore sequencing technology. We have developed a hybrid capture-based method to enrich pathogen DNA and 18 host mRNAs from patient blood samples collected in EDTA and PAXgene tubes respectively. We optimized the mRNA extraction by varying capture probe mix, probe design, lysis, binding, and elution conditions. The inter and intraday repeatability of this mRNA extraction was evaluated using four patient samples run across three days in replicates, using nanopore sequencing. After this evaluation, the preanalytics for mRNA extraction was integrated with the pre-established upstream pathogen DNA extraction. The resulting enriched nucleic acids were amplified by universal multiplex PCRs, pooled, and sequenced using Oxford Nanopore Technology. The real-time analysis of this integrated sequencing run was done using “SepsiProgDx” software, and reads were mapped against the proprietary database to identify pathogen/s and its AMR. In addition, a sepsis severity risk score for the same patient sample is determined by the integrated “Predict_d28” classifier algorithm that uses a weighted fuzzy-logic approach to predict mortality based on sepsis endotypes. We evaluated this integrated approach with 12 contrived Whole blood samples spiked with 50 CFU/mL of Staphylococcus aureus and Enterococcus faecium. Inter and Intraday repeatability: The trend analysis of relative gene expression profiles of host mRNA signatures revealed significant overlap across patient samples tested within and across three days, analyzed using Root mean square error metrics. Performance of Integrated workflow: We were able to detect Staphylococcus aureus and Enterococcus faecium at 50 CFU/mL, and derived host mRNA signature-based severity scores using the integrated method. The results of the integrated method were comparable to the individual pathogen identification (diagnostic) and Host response assays (prognostic). INSPIRE assay is a rapid, novel prognostic and diagnostic method that can identify high risk patients, infecting pathogen/s, and AMR determinants, directly from whole blood.