Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS 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 mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.

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.

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

Because of the widespread use of CT in the diagnosis of COVID 19, indeterminate presentations such as single, few or unilateral lesions amount to a considerable number. We aimed to develop a new classification and structured reporting system on CT imaging (COVID-19 S) that would facilitate the diagnosis of COVID-19 in the most accurate way. Our retrospective cohort included 803 patients with a chest CT scan upon suspicion of COVID 19. The patients' history, physical examination, CT findings, RT PCR, and other laboratory test results were reviewed, and a final diagnosis was made as COVID 19 or non-COVID 19. Chest CT scans were classified according to the COVID 19 S CT diagnosis criteria. Cohen's kappa analysis was used. Final clinical diagnosis was COVID-19 in 98 patients (12%). According to the COVID-19 S CT diagnosis criteria, the number of patients in the normal, compatible with COVID 19, indeterminate and alternative diagnosis groups were 581 (72.3%), 97 (12.1%), 16 (2.0%) and 109 (13.6%). When the indeterminate group was combined with the group compatible with COVID 19, the sensitivity and specificity of COVID-19 S were 99.0% and 87.1%, with 85.8% positive predictive value (PPV) and 99.1% negative predictive value (NPV). When the indeterminate group was combined with the alternative diagnosis group, the sensitivity and specificity of COVID-19 S were 93.9% and 96.0%, with 94.8% PPV and 95.2% NPV. COVID-19 S CT classification system may meet the needs of radiologists in distinguishing COVID-19 from pneumonia of other etiologies and help optimize patient management and disease control in this pandemic by the use of structured reporting.