Viral Metagenomics on Cerebrospinal Fluid

Abstract

Identifying the causative pathogen in central nervous system (CNS) infections is crucial for patient management and prognosis. Many viruses can cause CNS infections, yet screening for each individually is costly and time-consuming. Most metagenomic assays can theoretically detect all pathogens, but often fail to detect viruses because of their small genome and low viral load. Viral metagenomics overcomes this by enrichment of the viral genomic content in a sample. VIDISCA-NGS is one of the available workflows for viral metagenomics, which requires only a small input volume and allows multiplexing of multiple samples per run. The performance of VIDISCA-NGS was tested on 45 cerebrospinal fluid (CSF) samples from patients with suspected CNS infections in which a virus was identified and quantified by polymerase chain reaction. Eighteen were positive for an RNA virus, and 34 for a herpesvirus. VIDISCA-NGS detected all RNA viruses with a viral load >2 × 10⁴ RNA copies/mL (n = 6) and 8 of 12 of the remaining low load samples. Only one herpesvirus was identified by VIDISCA-NGS, however, when withholding a DNase treatment, 11 of 18 samples with a herpesvirus load >10⁴ DNA copies/mL were detected. Our results indicate that VIDISCA-NGS has the capacity to detect low load RNA viruses in CSF. Herpesvirus DNA in clinical samples is probably non-encapsidated and therefore difficult to detect by VIDISCA-NGS.

Keywords: CNS infection; VIDISCA-NGS; cerebrospinal fluid; encephalitis; metagenomics; metaviromics; next-generation sequencing; viromics; virus.

Figure 1

Detection of RNA viruses by virus discovery cDNA-AFLP (amplified fragment length polymorphism) next-generation sequencing (VIDISCA-NGS) in cerebrospinal fluid (CSF). Green dots: samples that were positive by VIDISCA-NGS for enterovirus, orange dots: samples that were positive by VIDISCA-NGS for HIV-1, white dots: samples that were negative by VIDISCA-NGS. The size of the dots corresponds to the number of viral reads. On the x-axis, the viral load in CSF is displayed; on the y-axis, the total number of sequence reads. Samples are divided into segments by a horizontal line at 15,000 reads and a vertical line at 2 × 10⁴ RNA copies/mL.

Figure 2

Background sequences in VIDISCA-NGS. Green dots: samples that were positive by VIDISCA-NGS, white dots: samples that were negative by VIDISCA-NGS, orange dots: the four samples containing an RNA virus not found by VIDISCA-NGS. On top the p-values are shown for the Mann-Whitney U test between the positive and negative VIDISCA-NGS samples. “Human” indicates human mitochondrial or genomic background, “Bacterial” indicates prokaryotic background, “Ambiguous” represents sequences with simultaneous hits to eukaryotes and prokaryotes, and “Unknown” are the sequences that do not match with any reference sequence.

Figure 3

Detection of herpesviruses by VIDISCA-NGS in CSF. The results of regular VIDISCA-NGS are in the left panel, results of DNase-free VIDICSA-NGS are in the right panel. If a sample contained multiple viruses, multiple data points are displayed for each of the co-infecting viruses. A vertical line is drawn to separate samples above and below 10⁴ DNA copies/mL. Green dots: samples that were positive by VIDISCA-NGS for HSV-1/2, blue dots: samples that were positive by VIDISCA-NGS for VZV, orange dots: samples that were positive by VIDISCA-NGS for CMV, white dots: samples that were negative by VIDISCA-NGS. The size of the dots corresponds to the number of viral reads. On the x-axis, the viral load in CSF is displayed; on the y-axis, the total number of sequence reads.

Figure 4

Effect of DNase on the detection of non-herpes DNA viruses by VIDISCA-NGS. On the x-axis, the viral species is displayed; on the y-axis, the total number of sequence reads. Left panel: Normal VIDISCA-NGS, right panel: DNase-free VIDSCA-NGS. Green dots: samples positive for the indicated virus, white dots: samples negative for the indicated virus. The size of the dots corresponds to the number of viral reads.

Figure 5

Effect of DNase on the detection of RNA and DNA viruses by VIDISCA-NGS in CSF. Viral read ratio (x-axis) is calculated as the ratio between the number of viral reads for samples with and without a DNase treatment, adjusted for the sequencing depth. Samples with a ratio >1 favor regular library preparation whereas samples with a ratio <1 favor a DNase-free treatment. Green dots: non-herpes DNA viruses, orange diamonds: herpesviruses, blue triangles: RNA viruses. On the y-axis, the viral species are displayed.