Exploring viral aetiology in upper respiratory tract infections: insights from metagenomic next-generation sequencing in Swiss outpatients before and during the SARS-CoV-2 pandemic.

AIMS OF THE STUDY: Upper respiratory tract infections are among the most common reasons for primary care consultations. They are diagnosed predominantly based on clinical assessment. Here, we investigated the benefit of viral metagenomic next-generation sequencing (mNGS) in an outpatient setting. METHODS: This prospective cross-sectional study included immunocompetent patients with acute upper respiratory tract infections. General practitioners collected pharyngeal swabs and demographic and clinical data. Specimens were analysed using viral mNGS and conventional tests. RESULTS: Two hundred seventy-seven patients were recruited by 21 general practitioners between 10/2019 and 12/2020, of which 91% had a suspected viral aetiology. For 138 patients (49.8%), mNGS identified one or more respiratory viruses. The mNGS showed a high overall agreement with conventional routine diagnostic tests. Rhinoviruses were the most frequently detected respiratory viruses (20.2% of patients). Viral mNGS reflected the influenza wave in early 2020 and the SARS-CoV-2 pandemic outbreak in Switzerland in March 2020. Notably, rhinoviruses continued to circulate despite non-pharmaceutical hygiene measures. CONCLUSIONS: Viral mNGS allowed the initial diagnosis to be retrospectively re-evaluated. Assuming reduced turnaround times, mNGS has the potential to directly guide the treatment of upper respiratory tract infections. On an epidemiological level, our study highlights the utility of mNGS in respiratory infection surveillance, allowing early detection of epidemics and providing information crucial for prevention.

How much should we sequence? An analysis of the Swiss SARS-CoV-2 surveillance effort.

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

Rapid and sensitive single-sample viral metagenomics using Nanopore Flongle sequencing.

The ability of viral metagenomic Next-Generation Sequencing (mNGS) to unbiasedly detect nucleic acids in a clinical sample is a powerful tool for advanced diagnosis of viral infections. When clinical symptoms do not provide a clear differential diagnosis, extensive laboratory testing with virus-specific PCR and serology can be replaced by a single viral mNGS analysis. However, widespread diagnostic use of viral mNGS is thus far limited by long sample-to-result times, as most protocols rely on Illumina sequencing, which provides high and accurate sequencing output but is time-consuming and expensive. Here, we describe the development of an mNGS protocol based on the more cost-effective Nanopore Flongle sequencing with decreased turnaround time and lower, yet sufficient sequencing output to provide sensitive virus detection. Sample preparation (6 h) and sequencing (2 h) times are substantially reduced compared to Illumina mNGS and allow detection of DNA/RNA viruses at low input (up to 33-38 cycle threshold of specific qPCR). Although Flongles yield lower sequencing output, direct comparison with Illumina mNGS on diverse clinical samples showed similar results. Collectively, the novel Nanopore mNGS approach is specifically tailored for use in clinical diagnostics and provides a rapid and cost-effective mNGS strategy for individual testing of severe cases.

Swiss public health measures associated with reduced SARS-CoV-2 transmission using genome data.

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020-the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures decoupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 to 98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred using a phylodynamic model. We found that transmission slowed 35 to 63% upon outbreak detection in summer 2020 but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.

Nosocomial COVID-19 Incidence and Secondary Attack Rates among Patients of Tertiary Care Center, Zurich, Switzerland.

Of 1,118 patients with COVID-19 at a university hospital in Switzerland during October 2020-June 2021, we found 83 (7.4%) had probable or definite healthcare-associated COVID-19. After in-hospital exposure, we estimated secondary attack rate at 23.3%. Transmission was associated with longer contact times and with lower cycle threshold values among index patients.

Antibodies from convalescent plasma promote SARS-CoV-2 clearance in individuals with and without endogenous antibody response.

BACKGROUNDNeutralizing antibodies are considered a key correlate of protection by current SARS-CoV-2 vaccines. The manner in which human infections respond to therapeutic SARS-CoV-2 antibodies, including convalescent plasma therapy, remains to be fully elucidated.METHODSWe conducted a proof-of-principle study of convalescent plasma therapy based on a phase I trial in 30 hospitalized COVID-19 patients with a median interval between onset of symptoms and first transfusion of 9 days (IQR, 7-11.8 days). Comprehensive longitudinal monitoring of the virological, serological, and disease status of recipients allowed deciphering of parameters on which plasma therapy efficacy depends.RESULTSIn this trial, convalescent plasma therapy was safe as evidenced by the absence of transfusion-related adverse events and low mortality (3.3%). Treatment with highly neutralizing plasma was significantly associated with faster virus clearance, as demonstrated by Kaplan-Meier analysis (P = 0.034) and confirmed in a parametric survival model including viral load and comorbidity (adjusted hazard ratio, 3.0; 95% CI, 1.1-8.1; P = 0.026). The onset of endogenous neutralization affected viral clearance, but even after adjustment for their pretransfusion endogenous neutralization status, recipients benefitted from plasma therapy with high neutralizing antibodies (hazard ratio, 3.5; 95% CI, 1.1-11; P = 0.034).CONCLUSIONOur data demonstrate a clear impact of exogenous antibody therapy on the rapid clearance of viremia before and after onset of the endogenous neutralizing response, and point beyond antibody-based interventions to critical laboratory parameters for improved evaluation of current and future SARS-CoV-2 therapies.TRIAL REGISTRATIONClinicalTrials.gov NCT04869072.FUNDINGThis study was funded via an Innovation Pool project by the University Hospital Zurich; the Swiss Red Cross Glückskette Corona Funding; Pandemiefonds of the UZH Foundation; and the Clinical Research Priority Program "Comprehensive Genomic Pathogen Detection" of the University of Zurich.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

SARS-CoV-2 variants reveal features critical for replication in primary human cells.

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure.

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.

Does respiratory co-infection facilitate dispersal of SARS-CoV-2? investigation of a super-spreading event in an open-space office.

BACKGROUND: Super-spreaders are individuals infecting disproportionately large numbers of contacts. They probably play a crucial role in the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We describe a super-spreading event within a team working in an open-space office and investigate factors potentially having facilitated SARS-CoV-2 transmission. METHODS: In this retrospective cohort study, semi-structured telephone interviews with all team members were carried out to identify symptoms, contacts, and adherence to basic hygiene measures. During site visits, we gathered information about workplace and seating arrangements. The secondary attack rate in office and households was calculated. Potential respiratory viral co-infections were assessed by multiplex PCR. SARS-CoV-2 whole-genome sequencing was performed using a tiled-amplicon sequencing approach. RESULTS: Of 13 team members, 11 fell ill with Coronavirus disease 2019 (COVID-19). Due to the sequence of events and full genome sequence data, one person was considered the index case for this outbreak, directly infecting 67 to 83% of the teammates. All team members reported repetitive close contacts among themselves during joint computer work, team meetings and a "Happy Birthday" serenade. Two individuals shared nuts and dates. The arrangement of the office and meeting rooms precluded sufficient adherence to physical distancing. The index case and a further individual were diagnosed with an adenovirus serotype 4 co-infection. CONCLUSION: We identified several environmental and behavioral factors that probably have facilitated the transmission of SARS-CoV-2. The relevance of the adenovirus co-infection remains unclear and merits further investigation.

Two Years of Viral Metagenomics in a Tertiary Diagnostics Unit: Evaluation of the First 105 Cases.

Metagenomic next-generation sequencing (mNGS) can capture the full spectrum of viral pathogens in a specimen and has the potential to become an all-in-one solution for virus diagnostics. To date, clinical application is still in an early phase and limitations remain. Here, we evaluated the impact of viral mNGS for cases analyzed over two years in a tertiary diagnostics unit. High throughput mNGS was performed upon request by the treating clinician in cases where the etiology of infection remained unknown or the initial differential diagnosis was very broad. The results were compared to conventional routine testing regarding outcome and workload. In total, 163 specimens from 105 patients were sequenced. The main sample types were cerebrospinal fluid (34%), blood (33%) and throat swabs (10%). In the majority of the cases, viral encephalitis/meningitis or respiratory infection was suspected. In parallel, conventional virus diagnostic tests were performed (mean 18.5 individually probed targets/patients). mNGS detected viruses in 34 cases (32%). While often confirmatory, in multiple cases, the identified viruses were not included in the selected routine diagnostic tests. Two years of mNGS in a tertiary diagnostics unit demonstrated the advantages of a single, untargeted approach for comprehensive, rapid and efficient virus diagnostics, confirming the utility of mNGS in complementing current routine tests.

Viral Metagenomics in the Clinical Realm: Lessons Learned from a Swiss-Wide Ring Trial.

Shotgun metagenomics using next generation sequencing (NGS) is a promising technique to analyze both DNA and RNA microbial material from patient samples. Mostly used in a research setting, it is now increasingly being used in the clinical realm as well, notably to support diagnosis of viral infections, thereby calling for quality control and the implementation of ring trials (RT) to benchmark pipelines and ensure comparable results. The Swiss NGS clinical virology community therefore decided to conduct a RT in 2018, in order to benchmark current metagenomic workflows used at Swiss clinical virology laboratories, and thereby contribute to the definition of common best practices. The RT consisted of two parts (increments), in order to disentangle the variability arising from the experimental compared to the bioinformatics parts of the laboratory pipeline. In addition, the RT was also designed to assess the impact of databases compared to bioinformatics algorithms on the final results, by asking participants to perform the bioinformatics analysis with a common database, in addition to using their own in-house database. Five laboratories participated in the RT (seven pipelines were tested). We observed that the algorithms had a stronger impact on the overall performance than the choice of the reference database. Our results also suggest that differences in sample preparation can lead to significant differences in the performance, and that laboratories should aim for at least 5-10 Mio reads per sample and use depth of coverage in addition to other interpretation metrics such as the percent of coverage. Performance was generally lower when increasing the number of viruses per sample. The lessons learned from this pilot study will be useful for the development of larger-scale RTs to serve as regular quality control tests for laboratories performing NGS analyses of viruses in a clinical setting.

Meningitis and epididymitis caused by Toscana virus infection imported to Switzerland diagnosed by metagenomic sequencing: a case report.

BACKGROUND: We report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland. CASE PRESENTATION: The patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days. CONCLUSIONS: This case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.

Metagenomic Virome Sequencing in Living Donor and Recipient Kidney Transplant Pairs Revealed JC Polyomavirus Transmission.

BACKGROUND: Before kidney transplantation, donors and recipients are routinely screened for viral pathogens using specific tests. Little is known about unrecognized viruses of the urinary tract that potentially result in transmission. Using an open metagenomic approach, we aimed to comprehensively assess virus transmission in living-donor kidney transplantation. METHODS: Living kidney donors and their corresponding recipients were enrolled at the time of transplantation. Follow-up study visits for recipients were scheduled 4-6 weeks and 1 year thereafter. At each visit, plasma and urine samples were collected and transplant recipients were evaluated for signs of infection or other transplant-related complications. For metagenomic analysis, samples were enriched for viruses, amplified by anchored random polymerase chain reaction (PCR), and sequenced using high-throughput metagenomic sequencing. Viruses detected by sequencing were confirmed using real-time PCR. RESULTS: We analyzed a total of 30 living kidney donor and recipient pairs, with a follow-up of at least 1 year. In addition to viruses commonly detected during routine post-transplant virus monitoring, metagenomic sequencing detected JC polyomavirus (JCPyV) in the urine of 7 donors and their corresponding recipients. Phylogenetic analysis confirmed infection with the donor strain in 6 cases, suggesting transmission from the transplant donor to the recipient, despite recipient seropositivity for JCPyV at the time of transplantation. CONCLUSIONS: Metagenomic sequencing identified frequent transmission of JCPyV from kidney transplant donors to recipients. Considering the high incidence rate, future studies within larger cohorts are needed to define the relevance of JCPyV infection and the donor's virome for transplant outcomes.

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples.

BACKGROUND: Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples. RESULTS: In order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs. CONCLUSIONS: The workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.