Metagenomic next-generation sequencing of cerebrospinal fluid for diagnosis of central nervous system infections: 7-year performance of a clinically validated test

Studies have shown that metagenomic next-generation sequencing (mNGS) testing of cerebrospinal fluid (CSF) in central nervous system (CNS) infections can improve diagnostic yields and provide actionable information. We analyzed the results of all CSF mNGS tests (n=4,828) performed at the University of California, San Francisco (UCSF) clinical microbiology laboratory from June 2016 to April 2023. We also assessed clinical metadata from a subset of samples that corresponded to a cohort of UCSF patients (n=1,164) who received CSF mNGS testing, and retrospectively evaluated performance compared to conventional microbiologic testing and adjudicated clinical diagnosis. Overall, 14.4% of CSF mNGS tests were positive for any microorganism. DNA viruses (7% of all samples) were detected most often, followed by RNA viruses (4.3%), bacteria (2.7%), fungi (1.4%), and parasites (0.5%). Using a composite gold standard obtained from clinical adjudication and all microbiological test results, sensitivity, specificity, and accuracy of CSF mNGS in the UCSF cohort who had clinically diagnosed infections were 56.5%, 98.8%, and 90.5%, respectively. The sensitivity of CSF mNGS testing (56.5%) was statistically higher than that from all direct detection testing from CSF (44.8%, p = 0.004), direct detection testing from samples other than CSF (15.2%, p<0.001), and indirect serologic testing (34%, p<0.001). When only considering diagnoses made by direct detection of pathogens on CSF, sensitivity of mNGS was 80.7%. These results justify the incorporation of CSF mNGS testing as part of the routine diagnostic workup in hospitalized patients presenting with potential CNS infection. ### Competing Interest Statement A.P.W., B.O., and S.M. are employed by and own equity in Delve Bio. C.Y.C. is a founder of Delve Bio and on the scientific advisory board for Delve Bio, Flightpath Biosciences, Biomeme, Mammoth Biosciences, BiomeSense and Poppy Health. He is also an inventor on US patent 11380421, 'Pathogen detection using next generation sequencing', under which algorithms for taxonomic classification, filtering and pathogen detection are used by SURPI+ software. C.Y.C. receives research support from Delve Bio and Abbott Laboratories, Inc. ### Funding Statement This work was funded, in part, by Delve Bio. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Retrospective patient chart review and analysis of patient clinical CSF results were performed under a biobanking protocol with waiver of consent approved by the UCSF Institutional Review Board. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data that support the findings of this study are available on request from the corresponding author C.Y.C.