Metagenomic sequencing in clinical virology: advances in pathogen detection and future prospects

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 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.LUMC / Geneeskund

Viral metagenomic sequencing in the diagnosis of meningoencephalitis: a review of technical advances and diagnostic yield

Introduction Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis, though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings; however, the exact diagnostic yield when used as a secondary test remains unknown. Areas covered This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective. Expert opinion The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Viral metagenomic sequencing in a cohort of international travellers returning with febrile illness

Background: 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.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Performance of five metagenomic classifiers for virus pathogen detection using respiratory samples from a clinical cohort

Viral metagenomics is increasingly applied in clinical diagnostic settings for detection of pathogenic viruses. While several benchmarking studies have been published on the use of metagenomic classifiers for abundance and diversity profiling of bacterial populations, studies on the comparative performance of the classifiers for virus pathogen detection are scarce. In this study, metagenomic data sets (n = 88) from a clinical cohort of patients with respiratory complaints were used for comparison of the performance of five taxonomic classifiers: Centrifuge, Clark, Kaiju, Kraken2, and Genome Detective. A total of 1144 positive and negative PCR results for a total of 13 respiratory viruses were used as gold standard. Sensitivity and specificity of these classifiers ranged from 83 to 100% and 90 to 99%, respectively, and was dependent on the classification level and data pre-processing. Exclusion of human reads generally resulted in increased specificity. Normalization of read counts for genome length resulted in a minor effect on overall performance, however it negatively affected the detection of targets with read counts around detection level. Correlation of sequence read counts with PCR Ct-values varied per classifier, data pre-processing (R-2 range 15.1-63.4%), and per virus, with outliers up to 3 log(10) reads magnitude beyond the predicted read count for viruses with high sequence diversity. In this benchmarking study, sensitivity and specificity were within the ranges of use for diagnostic practice when the cut-off for defining a positive result was considered per classifier.Molecular Epidemiolog

STROBE-metagenomics: a STROBE extension statement to guide the reporting of metagenomics studies

The term metagenomics refers to the use of sequencing methods to simultaneously identify genomic material from all organisms present in a sample, with the advantage of greater taxonomic resolution than culture or other methods. Applications include pathogen detection and discovery, species characterisation, antimicrobial resistance detection, virulence profiling, and study of the microbiome and microecological factors affecting health. However, metagenomics involves complex and multistep processes and there are important technical and methodological challenges that require careful consideration to support valid inference. We co-ordinated a multidisciplinary, international expert group to establish reporting guidelines that address specimen processing, nucleic acid extraction, sequencing platforms, bioinformatics considerations, quality assurance, limits of detection, power and sample size, confirmatory testing, causality criteria, cost, and ethical issues. The guidance recognises that metagenomics research requires pragmatism and caution in interpretation, and that this field is rapidly evolving.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

Introduction Exacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations. Objectives To study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS. Study design 88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006-2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations. Results By mNGS, 24/88 samples tested positive. Sensitivity and specificity, as compared with PCR, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). Additional viral pathogens detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral normalized species reads (log value) (p = 0.002). Patients with viral pathogens had lower percentages of bacteriophages (p<0.001). No correlation was found between viral reads and clinical markers. Conclusions The mNGS protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. Excellent negative predictive value implicates the power of mNGS to exclude any pathogenic respiratory viral infectious cause in one test, with consequences for clinical decision making. Reduced abundance of bacteriophages in COPD patients with viral pathogens implicates skewing of the virome during infection, with potential consequences for the bacterial populations, during infection

Recommendations for the introduction of metagenomic next-generation sequencing in clinical virology, part II: bioinformatic analysis and reporting

Metagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety of sample types from patients with infectious syndromes. mNGS is still in its early stages of broader translation into clinical applications. To further support the development, implementation, optimization and standardization of mNGS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mNGS for viral diagnostics to share methodologies and experiences, and to develop application guidelines. Following the ENNGS publication Recommendations for the introduction of mNGS in clinical virology, part I: wet lab procedure in this journal, the current manuscript aims to provide practical recommendations for the bioinformatic analysis of mNGS data and reporting of results to clinicians.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Metagenomic sequencing in clinical virology: advances in pathogen detection and future prospects

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 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.</p

Metagenomic sequencing in clinical virology: advances in pathogen detection and future prospects

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 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.</p

Coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness

Introduction: The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness.Aim: The aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness.Methods: The performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing SARS-CoV-2, SARS-CoV, and MERS-CoV, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery.Results: Classification of NGS reads using Centrifuge and Genome Detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. Low nucleotide and amino acid identity (81% and 84%, respectively, for SARS-CoV-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. Capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the SARS-CoV-2 genome, the latter increasing from 71% to 98%.Conclusion: The model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. The metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples