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

Karol Perlejewski,Marek Radkowski,Kamila Caraballo Cortѐs,Tomasz Laskus,Iwona Bukowska-Ośko,Marcin Paciorek,Małgorzata Rydzanicz,Agnieszka Pawełczyk,Sylwia Osuch,Tomasz Dzieciątkowski

doi:10.1038/s41598-020-73156-3

Karol Perlejewski, Marek Radkowski + Show 8 more

Open Access

https://doi.org/10.1038/s41598-020-73156-3

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Journal: Scientific Reports	Publication Date: Sep 30, 2020
Citations: 23	License type: open-access

Affiliation: Medical University of Warsaw

Abstract

Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined. Next-generation sequencing (NGS) based metagenomics has been successfully used to detect novel and rare infections, but its value for routine diagnosis of encephalitis remains unclear. The aim of the present study was to determine the sensitivity of shotgun metagenomic sequencing protocols, which include preamplification, and testing it against cerebrospinal fluid (CSF) samples from encephalitis patients. For sensitivity testing HIV and HBV positive sera were serially diluted in CSF from an uninfected patient. NGS repeatedly detected HIV and HBV sequences present at concentrations from 105 to 102 and from 105 to 10 viral copies/reaction, respectively. However, when the same protocols were applied to RT-PCR/PCR positive CSF samples from 6 patients with enteroviral encephalitis (median viral load 47 copies/ml) and 15 patients with HSV, CMV or VZV encephalitis (median viral load 148 copies/ml), only 7 (28.6%) were identified as positive. In conclusions, while NGS has the advantage of being able to identify a wide range of potential pathogens it seems to be less sensitive compared to the standard amplification-based assays in the diagnosis of encephalitis, where low viral loads are common.

Highlights

Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined
Diagnostics of central nervous system (CNS) infections is complicated by the sheer number of potential pathogens including over a hundred viruses as well as a larger number of bacteria, fungi and parasites which are capable of infecting CNS4, 5
In 2015 the U.S Food and Drug Administration (FDA) approved the first multiplex PCR system marketed by BioFire Diagnostics; USA which allows for detection of 14 different common pathogens but only seven viruses known to infect CNS are included in the testing p anel[8]

Summary

Introduction

Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined. Next-generation sequencing (NGS) based metagenomics has been successfully used to detect novel and rare infections, but its value for routine diagnosis of encephalitis remains unclear. The aim of the present study was to determine the sensitivity of shotgun metagenomic sequencing protocols, which include preamplification, and testing it against cerebrospinal fluid (CSF) samples from encephalitis patients. While NGS has the advantage of being able to identify a wide range of potential pathogens it seems to be less sensitive compared to the standard amplification-based assays in the diagnosis of encephalitis, where low viral loads are common. Technique is very robust for identification of low copy number pathogens, which is typical for viral encephalitis, it requires depletion of human/bacterial genetic background and enrichment of the target template[13,14]

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