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Genome Res. 2019 May;29(5):831-842. doi: 10.1101/gr.238170.118. Epub 2019 Apr 16.

Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid.

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Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California 94143, USA.
UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, California 94143, USA.
Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, California 90027, USA.
Department of Pediatrics, Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
Microsoft Research, Redmond, Washington 98052, USA.
Quest Diagnostics Nichols Institute, San Juan Capistrano, California 92675, USA.
Department of Pediatrics, Division of Pediatric Infectious Diseases, Children's National Health System, Washington, DC 20010, USA.
Department of Pediatrics, Microbiology, Immunology, and Tropical Medicine, The George Washington University School of Medicine, Washington, DC 20037, USA.
Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, San Francisco, California 94143, USA.
Contributed equally


Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.

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