Metagenomics enables the detection of all the genetic material of organisms present in a sample, making it a pathogen-agnostic approach for detecting common and rare or novel pathogens that are not... Show moreMetagenomics enables the detection of all the genetic material of organisms present in a sample, making it a pathogen-agnostic approach for detecting common and rare or novel pathogens that are not included in conventional testing. Beforehand, a clinician does not need to have a hypothesis of what pathogen is expected, unlike traditional polymerase chain reaction (PCR) testing.This thesis is focusing on diagnostic yield, clinical findings, and enhancing technical opportunities in viral metagenomics. The identification, typing, and quantification of viruses by means of viral metagenomics as a diagnostic tool are evaluated. Technical aspects are appraised for improved sensitivity and specificity of the wet and dry (bioinformatic) lab components of viral metagenomics. The use of a metagenomic protocol for virus discovery directly in a patient sample is assessed, and the best methods and approaches for performing genetic analysis of the SARS-CoV-2 virus are investigated.Viral metagenomic testing results in the identification of more viruses, therefore it is a valuable addition to current diagnostic test protocols. Additionally, it is a useful test for virus discovery and monitoring during infectious disease outbreaks caused by novel viruses. Show less
Background: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic... Show moreBackground: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyze the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness. Methods: Thirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods. Results: In 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected. Conclusions: Viral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing. Show less
