Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Air quality of health facilities in Spain.

The present study examines indoor air pollution in health facilities, focusing on compounds from various sources, such as industrial products, healthcare activities and building materials. It assesses chemical and microbiological concentrations in two public hospitals, two public healthcare centres, and one public health laboratory in Spain. Measurements included indoor air quality, microbiological contaminants, ambient parameters and non-target analysis across ten different locations. Outdoor air quality was also assessed in the surroundings of the hospitals. The results showed that around 350 substances were tentatively identified at a high confidence level, with over 50 % of compounds classified as of high toxicological risk. Three indoor and 26 outdoor compounds were fully confirmed with standards. These confirmed substances were linked to medical, industrial and agricultural activities. Indoor Air Quality (IAQ) results revealed that CO, CO2, formaldehyde (HCHO), O3 and total volatile organic compounds (TVOCs) showed average values above the recommended guideline levels in at least one of the evaluated locations. Moreover, maximum concentrations detected for CO, HCHO, O3 and TVOCs in hospitals surpassed those previously reported in the literature. SARS-CoV-2 was detected in three air environments, corresponding to COVID-19 patient areas. Fungi and bacteria concentrations were acceptable in all assessed locations, identifying different fungi genera, such as Penicillium, Cladosporium, Aspergillus, Alternaria and Botrytis.

Indoor Air Quality and Bioaerosols in Spanish University Classrooms.

A comprehensive study assessed indoor air quality parameters, focusing on relevant air pollutants such as particulate matter (PM10 and PM2.5), gaseous compounds (CO, CO2, formaldehyde, NO2) and volatile/semi-volatile organic chemicals, as well as respiratory viruses (including SARS-CoV-2), fungi and bacteria in Spanish university classrooms. Non-target screening strategies evaluated the presence of organic pollutants inside and outside the classrooms. Saliva samples from teachers and students were collected to explore correlations between respiratory viruses in the air and biological samples. Indoor results revealed the punctual exceedance of recommended guidelines for CO2, formaldehyde (HCHO), volatile organic compounds (TVOCs) and PM in the least naturally ventilated classrooms. Significant differences occurred between the classes, with the least ventilated one showing higher average concentrations of CO2, HCHO, NO2, PM10 and PM2.5. A respiratory virus (rhinovirus/enterovirus) was detected in the medium naturally ventilated classroom, although saliva samples tested negative. Suspect screening tentatively identified 65 substances indoors and over 200 outdoors, with approximately half reporting a high toxicological risk based on the Cramer rules. The study provides a comprehensive overview of indoor air quality, respiratory viruses and organic pollutants in university classrooms, highlighting the variations and potential health risks associated with ventilation differences.

Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

Early estimates of nirsevimab immunoprophylaxis effectiveness against hospital admission for respiratory syncytial virus lower respiratory tract infections in infants, Spain, October 2023 to January 2024.

The monoclonal antibody nirsevimab was at least 70% effective in preventing hospitalisations in infants with lower respiratory tract infections (LRTI) positive for respiratory syncytial virus (RSV) in Spain (Oct 2023-Jan 2024), where a universal immunisation programme began late September (coverage range: 79-99%). High protection was confirmed by two methodological designs (screening and test-negative) in a multicentre active surveillance in nine hospitals in three regions. No protection against RSV-negative LRTI-hospitalisations was shown. These interim results could guide public-health decision-making.

Development of Photonic Multi-Sensing Systems Based on Molecular Gates Biorecognition and Plasmonic Sensors: The PHOTONGATE Project.

This paper presents the concept of a novel adaptable sensing solution currently being developed under the EU Commission-founded PHOTONGATE project. This concept will allow for the quantification of multiple analytes of the same or different nature (chemicals, metals, bacteria, etc.) in a single test with levels of sensitivity and selectivity at/or over those offered by current solutions. PHOTONGATE relies on two core technologies: a biochemical technology (molecular gates), which will confer the specificity and, therefore, the capability to be adaptable to the analyte of interest, and which, combined with porous substrates, will increase the sensitivity, and a photonic technology based on localized surface plasmonic resonance (LSPR) structures that serve as transducers for light interaction. Both technologies are in the micron range, facilitating the integration of multiple sensors within a small area (mm2). The concept will be developed for its application in health diagnosis and food safety sectors. It is thought of as an easy-to-use modular concept, which will consist of the sensing module, mainly of a microfluidics cartridge that will house the photonic sensor, and a platform for fluidic handling, optical interrogation, and signal processing. The platform will include a new optical concept, which is fully European Union Made, avoiding optical fibers and expensive optical components.

In vivo effect of mouthwashes on viable viral load of SARS-CoV-2 in saliva: a pilot study.

Current data on the efficacy of antiseptic mouthwashes to reduce viral load are contradictory. Firstly, in vitro data indicate very strong virucidal effects that are not replicated in clinical studies. Secondly, most clinical studies identify a limited effect, do not include a control/placebo group, or do not evaluate viral viability in an infection model. In the current manuscript, we perform a double-blind, randomized clinical trial where salivary viral load was measured before and after the mouthwash, and where saliva samples were also cultured in an in vitro infection model of SARS-CoV-2 to evaluate the effect of mouthwashes on viral viability. Our data show a 90-99% reduction in SARS-CoV-2 salivary copies with one of the tested mouthwashes, although we show that the remaining viruses are mostly viable. In addition, our data suggest that the active ingredient concentration and the overall excipients' formulation can play an important role; and most importantly, they indicate that the effect is not immediate, being significant at 15 min and having maximum effectiveness after 1 h. Thus, we show that some oral mouthwashes can be useful in reducing viral transmission, although their efficacy must be improved through refined formulations or revised protocols.

Effect of oral antiseptics in reducing SARS-CoV-2 infectivity: evidence from a randomized double-blind clinical trial.

ABSTRACTBackground: In vitro studies have shown that several oral antiseptics have virucidal activity against SARS-CoV-2. Thus, mouthwashes have been proposed as an easy to implement strategy to reduce viral transmission. However, there are no data measuring SARS-CoV-2 viability after mouthwashes in vivo. METHODS: In this randomized double-blind, five-parallel-group, placebo-controlled clinical trial, SARS-CoV-2 salivary viral load (by quantitative PCR) and its infectious capacity (incubating saliva in cell cultures) have been evaluated before and after four different antiseptic mouthwashes and placebo in 54 COVID-19 patients. RESULTS: Contrary to in vitro evidence, salivary viral load was not affected by any of the four tested mouthwashes. Viral culture indicated that cetylpyridinium chloride (CPC) significantly reduced viral infectivity, but only at 1-hour post-mouthwash. CONCLUSION: These results indicate that some of the mouthwashes currently used to reduce viral infectivity are not efficient in vivo and, furthermore, that this effect is not immediate, generating a false sense of security.Trial registration: ClinicalTrials.gov identifier: NCT04707742..

Clinical and phylogenetic influenza dynamics for the 2019-20 season in the global influenza hospital surveillance network (GIHSN) - Pilot study.

BACKGROUND: The Global Influenza Hospital Surveillance Network (GIHSN) has operated with the aim of investigating epidemiological and clinical factors related to severe influenza-related hospitalisations. STUDY DESIGN: A common GIHSN core protocol for prospective patient enrolment was implemented. Hospital personnel completed a standardized questionnaire regarding the included patients' medical history, compiled a hospitalisation summary, collected an upper respiratory swab sample for laboratory diagnosis, and genome sequencing was performed for a subset of samples. Patient data were compared according to influenza subtype, lineage, and phylogenetic groups using the Fisher's exact test. RESULTS: From September 2019 to May 2020, 8791 patients aged ≥5 years were included. Among them, 3021 (34.4%) had a laboratory-confirmed influenza diagnosis. Influenza A(H1N1)pdm09 dominated the season among all age groups, while the B/Victoria-like lineage accounted for over half of the infections among younger age groups (5-49 years). Sequencing of the hemagglutinin segment was possible for 623 samples and revealed an influenza A and B clade frequency among severe influenza hospitalisations similar to other medically attended surveillance networks, such as the WHO GISRS. No phylogenetic clustering was observed among hemagglutinin substitutions depending on the administration of supplemental oxygen or vaccine failure. CONCLUSIONS: The GIHSN confirms its ability as an international hospital-based active surveillance network to provide valuable information on influenza infection dynamics in hospital settings. Increasing the number of participating sites and compiling more complete data, such as genome sequencing, will allow the exploration of associations between viral factors, vaccine protection, and disease severity.

Retrospective screening for SARS-CoV-2 among influenza-like illness hospitalizations: 2018-2019 and 2019-2020 seasons, Valencia region, Spain.

On 9 March 2020, the World Health Organization (WHO) Global Influenza Programme (GIP) asked participant sites on the Global Influenza Hospital Surveillance Network (GIHSN) to contribute to data collection concerning severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We re-analysed 5833 viral RNA archived samples collected prospectively from hospital admissions for influenza-like illness (ILI) in the Valencia Region of Spain by the Valencia Hospital Surveillance Network for the Study of Influenza and Other Respiratory Viruses (VAHNSI) network (four hospitals, catchment area population 1 118 732) during the pre-pandemic 2018/2019 (n = 4010) and pandemic 2019/2020 (n = 1823) influenza seasons for the presence of SARS-CoV-2. We did not find evidence for community-acquired SARS-CoV-2 infection in hospital admissions for ILI in our region before early March 2020.

Role of age and birth month in infants hospitalized with RSV-confirmed disease in the Valencia Region, Spain.

BACKGROUND: RSV is the leading cause of hospital admissions in infants and the principal cause of bronchiolitis in young children. There is a lack of granular data on RSV-associated hospitalization per season using laboratory confirmed results. Our current study addresses this issue and intends to fill this gap. METHODS: The study was conducted from 2014 through 2018, in 4 to 10 hospitals in the Valencia Region, Spain. Infants included in this study were admitted in hospital through the Emergency Department with a respiratory complaint and tested by RT-PCR for RSV in a central laboratory. RESULTS: Incidence rates of RSV-associated hospitalization varied by season and hospital. Overall, the highest incidence rates were observed in 2017/2018. RSV-associated hospitalization was highest in infants below 3 months of age and in those born before or at the beginning of the RSV season. Almost 54% of total infants hospitalized with laboratory confirmed RSV were found to be born outside the season, from April to October. The RSV positivity rate by ICD-10 discharged codes varied by season and age with results from 48% to 57% among LRI (J09-J22). CONCLUSION: The study was instrumental in bringing forth the time unpredictability of RSV epidemics, the critical impact of age, and the comparable distribution of RSV-associated hospitalization in infants born on either side of the RSV season. These data could help in better characterization of the population that drives the healthcare burden and is crucial for the development of future immunization strategies, especially with upcoming vaccines in against RSV.

Cytomegalovirus specific polyfunctional T-cell responses expressing CD107a predict control of CMV infection after liver transplantation.

Cytomegalovirus (CMV) viral load after liver transplantation (LT) is controlled by cell mediated immune responses (CMI). Quantification of CMV-specific T-cells may identify patients who control CMV spontaneously and avoid expensive and potentially toxic antiviral therapies. Prospective post-LT clinical, virological and immunological monitoring was carried out up to 1-year post-LT in a cohort of adult recipients. The CMV-specific T-cell response was characterized using flow cytometry intracellular cytokine staining in 49 LT recipients-R (79.6% R+, 20.4% R-). CMV infection occurred in 24 patients (18 D+/R+ and 6 D+/R-). Only patients with undetectable polyfunctional CMV-specific CD4+ T-cells developed CMV infection. Predictive models showed that polyfunctional CMV-specific CD4+ T-cells pre-existing before LT are protective for CMV reactivation posttransplantation. Quantitation of CD4+ T-cell responses to CMV may be a useful marker for spontaneous control of viral replication to tailor antiviral prophylaxis after LT.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Indoor Air Quality including Respiratory Viruses.

The detection of SARS-CoV-2 in indoor environments is a cause of increasing concern. In this study, three sampling methodologies have been used in order to collect SARS-CoV-2 and 17 other respiratory viruses in indoor air, combined with a new analytical process to analyze respiratory viruses. Different areas of an ophthalmological hospital were investigated for the presence of these airborne viruses. Moreover, indoor air quality (IAQ) parameters (carbon dioxide, CO2; carbon monoxide, CO; nitrogen dioxide, NO2; volatile organic compounds, VOCs; formaldehyde, HCHO; and particulate matter, PM) have been examined to study the relationship between IAQ and airborne viruses. All indoor air and surface samples assessed were found to be negative for SARS-CoV-2. Nevertheless, another airborne respiratory virus (HRV/ENV) was detected, illustrating that the methodology set out here is a suitable one. Regarding the results for the IAQ, chemical parameters studied in the hall and waiting room of the hospital presented acceptable values. However, in the doctor's consultation room VOCs and HCHO show some instantaneous levels higher than the recommended guide values. The methodological approach described in this paper, integrating conventional IAQ and the assessment of bioaerosols, can be used in research and control programs aimed at promoting a healthy indoor environment.

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.

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.

Whole-Genome Analysis Surveillance of Influenza A Virus Resistance to Polymerase Complex Inhibitors in Eastern Spain from 2016 to 2019.

Molecular surveillance by whole-genome sequencing was used to monitor the susceptibility of circulating influenza A viruses to three polymerase complex inhibitors. A total of 12 resistance substitutions were found among 285 genomes analyzed, but none were associated with high levels of resistance. Natural resistance to these influenza A antivirals is currently uncommon.

Influenza Vaccine Effectiveness and Waning Effect in Hospitalized Older Adults. Valencia Region, Spain, 2018/2019 Season.

Influenza vaccination is annually recommended for specific populations at risk, such as older adults. We estimated the 2018/2019 influenza vaccine effectiveness (IVE) overall, by influenza subtype, type of vaccine, and by time elapsed since vaccination among subjects 65 years old or over in a multicenter prospective study in the Valencia Hospital Surveillance Network for the Study of Influenza and other Respiratory Viruses (VAHNSI, Spain). Information about potential confounders was obtained from clinical registries and/or by interviewing patients and vaccination details were only ascertained by registries. A test-negative design was performed in order to estimate IVE. As a result, IVE was estimated at 46% (95% confidence interval (CI): (16%, 66%)), 41% (95% CI: (-34%, 74%)), and 45% (95% CI: (7%, 67%)) against overall influenza, A(H1N1)pdm09 and A(H3N2), respectively. An intra-seasonal not relevant waning effect was detected. The IVE for the adjuvanted vaccine in ≥75 years old was 45% (2%, 69%) and for the non-adjuvanted vaccine in 65-74 years old was 59% (-16%, 86%). Thus, our data revealed moderate vaccine effectiveness against influenza A(H3N2) and not significant against A(H1N1)pdm09. Significant protection was conferred by the adjuvanted vaccine to patients ≥75 years old. Moreover, an intra-seasonal not relevant waning effect was detected, and a not significant IVE decreasing trend was observed over time.

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.

A population-based screening for hepatitis C antibodies and active infection using a point-of-care test in a low prevalence area.

BACKGROUND: Data on the true prevalence of hepatitis C virus (HCV) infection in the general population is essential. We evaluated a program implementing free universal HCV screening using a non-invasive point-of-care test (POCT) (OraQuick-HCV rapid test) in oral fluid in an urban area in Valencia, South-Eastern Spain. METHODS: A cross-sectional study was performed during 2015-2017. Free HCV screening was offered by regular mail to 11,500 individuals aged 18 and over, randomly selected from all census residents in the Health Department. All responding participants filled in a questionnaire about HCV infection risk factors and were tested in their tertiary Hospital. In those with a positive POCT, results were confirmed by enzyme-immunoassay and HCV-RNA. RESULTS: 1,206 persons agreed to participate (response rate: 11.16%). HCV antibodies were detected in 19 (1.60%) cases (age-sex standardized rate: 1.31%; 95%CI: 0.82-2.07), but only 8 showed positive HCV-RNA (age-sex standardized rate: 0.56%; 95%CI: 0.28-1.14). The majority (89%) of the cases were born before 1965 and 74% had at least one known risk factor for HCV infection. All anti-HCV positive individuals were already aware of their infection, and no undiagnosed cases were detected. The performance of the POCT was excellent for detecting active infection. CONCLUSIONS: These preliminary data suggest that HCV population screening with a POCT is feasible but, in our setting, mailing recruiting is not effective (11% response rate). The low prevalence of HCV antibodies and active infection in the participant population (with no new diagnoses made) suggests that, in our setting, underdiagnosis may be uncommon.