Laboratory Validation of a Clinical Metagenomic Sequencing Assay for Pathogen Detection in Cerebrospinal Fluid

Abstract

6Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been 3 7 successfully tested in proof-of-concept case studies in patients with acute illness of unknown 3 8 etiology, but to date has been largely confined to research settings. Here we developed and 3 9 validated an mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from 4 0 cerebrospinal fluid (CSF) in a licensed clinical laboratory. A clinical bioinformatics pipeline, 4 1 SURPI+, was developed to rapidly analyze mNGS data, automatically report detected 4 2 pathogens, and provide a graphical user interface for evaluating and interpreting results. We 4 3 established quality metrics, threshold values, and limits of detection of between 0.16 -313 4 4 genomic copies or colony forming units per milliliter for each representative organism type. 4 5 Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, a spiked 4 6 phage used as an internal control was a reliable indicator of sensitivity loss. Diagnostic test 4 7 accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% 4 8 sensitivity and 99% specificity compared to original clinical test results, with 81% positive 4 9

Clinical metagenomics

“…In laboratory test development, well-characterized, standardized and validated reference materials or databases that enable measurement of performance characteristics of an assay, including sensitivity, specificity and accuracy. The details for the specific steps vary by laboratory and are described extensively elsewhere [33][34][35][36][37] .…”

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance

“…To assess whether metagenomic detection sensitivity for off-target DNA viral and non-viral pathogens was retained using MSSPE, we evaluated the method using a representative mixture of seven organisms developed as a standardized positive control for a clinical metagenomic assay from CSF 33 . The normalized reads per million (RPM) corresponding to each of the seven pathogen types were comparable when using the ArboV or HFV spiked primer panel versus RH primers alone ( Supplementary Table 10).…”

“…Sequencing data from Illumina MiSeq or HiSeq instruments were analysed for viruses using the sequence-based ultra-rapid pathogen identification (SURPI+ v.1.0) computational pipeline (UCSF), a modified version of a previously published bioinformatics analysis pipeline for pathogen identification from mNGS sequence data 33,50 . Specifically, the SURPI+ pipeline modifications include: updated reference databases based on the NCBI nucleotide database (March 2015 build); a filtering algorithm for the exclusion of false-positive hits from database misannotations; and taxonomic classification for species-level identification.…”

“…Specifically, the SURPI+ pipeline modifications include: updated reference databases based on the NCBI nucleotide database (March 2015 build); a filtering algorithm for the exclusion of false-positive hits from database misannotations; and taxonomic classification for species-level identification. After SURPI+ identification of viral reads, viral reads were trimmed by 13 nucleotides at the 5′ and 3′ end to remove the spiked primers before mapping to the most closely matched reference genomes and visualization using SURPI+ 33,50 or Geneious 11.1.3 (ref. 51 ).…”

Advanced Immunotechnological Methods for Detection and Diagnosis of Viral Infections: Current Applications and Future Challenges

“…It is noteworthy that metagenomic next-generation sequencing assay (mNGS) for pan-pathogen detection has been effectively tested in patients with acute illness of several viral etiologies. In this connection a customized bioinformatics pipeline, SURPI+, was recently designed to quickly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results (Miller et al 2019). Panpathogen Metagenomic sequencing assay for SLEV (St. Louis encephalitis virus) infection in CSF (cerebrospinal fluid) is an unbiased approach to infectious disease testing, although several challenges still remain relating to test availability, interpretation, and validation that were reported.…”