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.

Next-generation sequencing in the diagnosis of viral encephalitis: sensitivity and clinical limitations

“…In analogous experiments with HIV spiked into CSF free matrix Schlaberg et al found the sensitivity of metagenomics to be approximately 100 copies/ml 26 . Similar sensitivity was reported by Edridge et al 27 who used virus discovery cDNA amplified fragment length polymorphism-next-generation sequencing protocol (VIDICSA-NGS) for the detection of RNA viruses in CSF. The authors were able to detect HIV in a sample containing 1.07 × 10 2 viral copies/ml.…”

“…In our previous study we found that while DNase treatment resulted in more than twofold decrease in the number of host-derived sequences and increased the number of bacterial and other sequences 30-50 times, it reduced the yield of HHV-1 four-fold and markedly lowered gene coverage when plotted to full-length HHV-1 reference sequence 30 . This sensitivity of HHV-1 to DNase treatment has been since confirmed by others 27 and seems to be due to the fact that in cell-free clinical material DNA of Herpesviruses is largely present in highly fragmented naked form and not as encapsulated virions 31 . In the study by Hong et al 32 which did not use the DNase digestion step, metagenomics detected HHV-1 in 5 out of 7 RT-PCR positive patients.…”

Human Coronaviruses and Other Respiratory Viruses: Underestimated Opportunistic Pathogens of the Central Nervous System?

“…A fast and accurate diagnosis would certainly improve prognosis for patients with a suspected CNS infection. Identification of a specific virus provides relevant information on how to treat a patient; therefore, the development of modern technologies, such as high throughput sequencing (Next Generation Sequencing) are warranted as it represents a potentially unbiased marvelous tool for rapid and robust diagnosis of unexplained encephalitis or other types of encephalopathies or neuronal manifestations, especially in the context where more traditional techniques have failed to identify the etiological agent [21,108,111,244,321,322]. Therefore, although our attention is mainly on a few different viruses such as HSV, arboviruses and enteroviruses, it may now be the time to look at CNS viral infection from another perspective.…”

“…Including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods [8,70,[108][109][110]. However, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of CNS viral infections often remains unknown.…”

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

“…However, despite our two-pronged RNA and DNA approach with a preamplication step no eukaryotic viruses were detected even though three of the analyzed samples were positive for Herpesviruses by specific real-time PCR. 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 ].…”