RT Journal
A1 Miller, Steve
A1 Naccache, Samia N.
A1 Samayoa, Erik
A1 Messacar, Kevin
A1 Arevalo, Shaun
A1 Federman, Scot
A1 Stryke, Doug
A1 Pham, Elizabeth
A1 Fung, Becky
A1 Bolosky, William J.
A1 Ingebrigtsen, Danielle
A1 Lorizio, Walter
A1 Paff, Sandra M.
A1 Leake, John A.
A1 Pesano, Rick
A1 DeBiasi, Roberta
A1 Dominguez, Samuel
A1 Chiu, Charles Y.
T1 Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid
JF Genome Research 
JO Genome Research 
YR 2019 
FD May 01 
VO 29 
IS 5 
SP 831 
OP 842 
DO 10.1101/gr.238170.118 
UL http://genome.cshlp.org/content/29/5/831.abstract 
AB 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.