title: 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 102HIV copies and 103HSV 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.
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Bukowska-Ośko
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland. karol.perlejewski@wum.edu.pl.
authors.forename: Karol
authors.initials: K
authors.lastname: Perlejewski
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Motooka
authors.affiliation: Department of Clinical Science, Bergen University, 5021, Bergen, Norway.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Stokowy
authors.affiliation: Department of the Medical Genetics, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kosińska
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Popiel
authors.affiliation: Department of the Medical Genetics, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Rafał
authors.initials: R
authors.lastname: Płoski
authors.affiliation: Municipal Hospital for Infectious Diseases, 37 Wolska St, 01-201, Warsaw, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Horban
authors.affiliation: Municipal Hospital for Infectious Diseases, 37 Wolska St, 01-201, Warsaw, Poland.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Lipowski
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Kamila
authors.initials: K
authors.lastname: Caraballo Cortés
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Pawełczyk
authors.affiliation: Department of Laboratory Medicine and Clinical Immunology of Developmental Age, Warsaw Medical University, 24 Marszałkowska St, 00-576, Warsaw, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Demkow
authors.affiliation: Department of Neurology, Military Institute of Medicine, 128 Szaserów St, 04-141, Warsaw, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Stępień
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Radkowski
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Warsaw Medical University, 3C Pawińskiego St, 02-106, Warsaw, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Laskus
keywords: Bacteria; Cerebrospinal fluid; DNA; Encephalitis; Metagenomics analysis; Next-generation sequencing; Pathogens; Viruses