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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.

Author information

1
Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California 94143, USA.
2
UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, California 94143, USA.
3
Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, California 90027, USA.
4
Department of Pediatrics, Children's Hospital Colorado and University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
5
Microsoft Research, Redmond, Washington 98052, USA.
6
Quest Diagnostics Nichols Institute, San Juan Capistrano, California 92675, USA.
7
Department of Pediatrics, Division of Pediatric Infectious Diseases, Children's National Health System, Washington, DC 20010, USA.
8
Department of Pediatrics, Microbiology, Immunology, and Tropical Medicine, The George Washington University School of Medicine, Washington, DC 20037, USA.
9
Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, San Francisco, California 94143, USA.
#
Contributed equally

Abstract

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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