Development and validation of a sample-to-answer metagenomics workflow for comprehensive pathogen detection in central nervous system infections

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ABSTRACT This research will ultimately advance human health by improving the diagnosis and treatment of central nervous system infections including meningitis and encephalitis, where the causative pathogen is not identified in up to 50% of cases. These infections are associated with significant morbidity and mortality; therefore, rapid and accurate diagnosis is crucial for appropriate patient management and to improve outcomes. Unfortunately, due to the variety of organisms that can cause central nervous system infections, current standard of care diagnostic methods suffer from long turnaround times and high false negative rates. Metagenomic next- generation sequencing (mNGS) has emerged as a promising universal detection method for infectious disease, enabling the simultaneous identification and characterization of viral, bacterial, and fungal pathogens directly from the DNA/RNA present in clinical samples. However, despite the increasing adoption of molecular diagnostics in clinical microbiology labs, mNGS has not become widespread due to its cost, turnaround time, and the technical expertise required to produce and analyze the data. The product we are developing addresses all of these obstacles. Galileo ONE – CSF is a sample-to-answer platform that includes all of the reagents, controls, protocols, and software required to detect and report clinically relevant pathogens directly from a cerebrospinal fluid (CSF) sample in ~24 hours. This platform incorporates several proprietary technologies, including a library preparation module that reduces abundant human sequences and a quantitative bioinformatics pipeline that increases sensitivity for sequence variants. This all-in-one bundle allows labs to easily evaluate and adopt mNGS in their own laboratories without the need for extensive genomics or bioinformatics expertise. During our Phase I-equivalent work, we used contrived samples to assess the feasibility of using CSF as a sample type; the results show that we can generate high-quality libraries and detect a representative set of pathogens in CSF using our Galileo ONE workflow, which was initially validated on plasma. Therefore, we are ready to move into the development phase of the product development process. In Phase II, we will perform analytical and preliminary clinical validation of the Galileo ONE – CSF platform, complete development of CSF-specific kit controls, and perform beta testing on contrived and clinical samples with our collaborators. This product will be the first deployed mNGS platform validated for CSF, enabling us to take advantage of the growing market for NGS-based infectious disease diagnostics, which is expected to reach $2.1B by 2027. Galileo ONE – CSF will empower end users, who include researchers, reference laboratories, and pharma/biotech partners, to adopt metagenomics to address the current gap in tools for detecting central nervous system infections.