Sensitivity of Next-Generation Sequencing Metagenomic Analysis for Detection of RNA and DNA Viruses in Cerebrospinal Fluid: The Confounding Effect of Background Contamination

Abstract

Next-generation sequencing (NGS) followed by metagenomic enables the detection and identification of known as well as novel pathogens. It could be potentially useful in the diagnosis of encephalitis, caused by a variety of microorganisms. The aim of the present study was to evaluate the sensitivity of isothermal RNA amplification (Ribo-SPIA) followed by NGS metagenomic analysis in the detection of human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cerebrospinal fluid (CSF). Moreover, we analyzed the contamination background. We detected 10 HIV copies and 10 HSV copies. The analysis of control samples (two water samples and one CSF sample from an uninfected patient) revealed the presence of human DNA in the CSF sample (91 % of all reads), while the dominating sequences in water were qualified as 'other', related to plants, plant viruses, and synthetic constructs, and constituted 31 % and 60 % of all reads. Bacterial sequences represented 5.9 % and 21.4 % of all reads in water samples and 2.3 % in the control CSF sample. The bacterial sequences corresponded mainly to Psychrobacter, Acinetobacter, and Corynebacterium genera. In conclusion, Ribo-SPIA amplification followed by NGS metagenomic analysis is sensitive for detection of RNA and DNA viruses. Contamination seems common and thus the results should be confirmed by other independent methods such as RT-PCR and PCR. Despite these reservations, NGS seems to be a promising method for the diagnosis of viral infections.

Search for viral agents in cerebrospinal fluid in patients with multiple sclerosis using real-time PCR and metagenomics

“…This discrepancy could be due to the fact that metagenomic workflows are less sensitive than specific real time RT-PCR/PCR assays and thus may fail in analysis of low viral-copy CSF samples [ 52 ]. Using serial dilutions of HIV and HSV positive sera in negative CSF, we have previously found that the limit of detection was 10 2 and 10 3 copies per reaction, respectively, while in the current study viral load in metagenomics-negative real-time PCR-positive samples ranged from 550 to 1650 copies/ml [ 53 ].…”

“…These mostly represented phage species and artifact sequences, which are particularly common for samples with a low DNA and RNA loads [ 54 , 55 ]. Moreover, the reagents themselves could be the source of contaminating foreign sequences and may affect the interpretation of metagenomic results [ 53 , 56 ].…”

Diagnostic Value of Next-Generation Sequencing to Detect Periprosthetic Joint Infection

“…The vast majority of sequences (usually > 99%) come from human hosts, consequently limiting the overall analytical sensitivity of pathogen detection methods [33] . Another potential challenge is that testing samples, reagents, or contaminating microorganisms in the laboratory environment can affect the accuracy of results [34,35] . In addition, while metagenes can be used to detect pathogenic bacteria, they cannot be used for drug sensitivity tests at the same time, which remains a persistent issue.…”

A Collaborative Tale of Diagnosing and Treating Chronic Pulmonary Aspergillosis, from the Perspectives of Clinical Microbiologists, Surgical Pathologists, and Infectious Disease Clinicians

“…One major downside to shotgun metagenomics is the interference with human DNA and environmental contaminants [89][90][91][92]. Specimen pre-processing can increase pathogen-to-human DNA ratio, allowing for increased pathogen detection sensitivity and reduced human sequences [93].…”