The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance ...of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.
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.
To study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rhinovirus infections occur frequently throughout life and have been reported in about one-third of asymptomatic cases. The clinical significance of sequential rhinovirus infections remains unclear. ...To determine the incidence and clinical relevance of sequential rhinovirus detections, nasopharyngeal samples from 2485 adults with acute cough/lower respiratory illness were analysed. Patients were enrolled prospectively by general practitioners from 12 European Union countries during three consecutive years (2007-2010). Nasopharyngeal samples were collected at the initial general practitioner consultation and 28 days thereafter and symptom scores were recorded by patients over that period. Rhinovirus RNA was detected in 444 (18%) out of 2485 visit one samples and in 110 (4.4%) out of 2485 visit two respiratory samples. 21 (5%) of the 444 patients had both samples positive for rhinovirus. Genotyping of both virus detections was successful for 17 (81%) out of 21 of these patients. Prolonged rhinovirus shedding occurred in six (35%) out of 21 and re-infection with a different rhinovirus in 11 (65%) out of 21. Rhinovirus re-infections were significantly associated with chronic obstructive pulmonary disease (p=0.04) and asthma (p=0.02) and appeared to be more severe than prolonged infections. Our findings indicate that in immunocompetent adults rhinovirus re-infections are more common than prolonged infections, and chronic airway comorbidities might predispose to more frequent rhinovirus re-infections.
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.
Differences in clinical impact between rhinovirus (RVs) species and types in adults are not well established. The objective of this study was to determine the epidemiology and clinical impact of the ...different RV species.
We conducted a prospective study of RVs infections in adults with acute cough/lower respiratory tract infection (LRTI) and asymptomatic controls. Subjects were recruited from 16 primary care networks located in 11 European countries between 2007 and 2010. RV detection and genotyping was performed by means of real time and conventional reverse-transcriptase polymerase chain reaction assays, followed by sequence analysis. Clinical data were obtained from medical records and patient symptom diaries.
RVs were detected in 566 (19%) of 3016 symptomatic adults, 102 (4%) of their 2539 follow-up samples and 67 (4%) of 1677 asymptomatic controls. Genotyping was successful for 538 (95%) symptomatic subjects, 86 (84%) follow-up infections and 62 (93%) controls. RV-A was the prevailing species, associated with an increased risk of LRTI as compared with RV-B (relative risk (RR), 4.5; 95% CI 2.5 to 7.9; p<0.001) and RV-C (RR 2.2; 95% CI 1.2 to 3.9; p=0.010). In symptomatic subjects, RV-A loads were higher than those of RV-B (p=0.015). Symptom scores and duration were similar across species. More RV-A infected patients felt generally unwell in comparison to RV-C (p=0·023). Of the 140 RV types identified, five were new types; asymptomatic infections were associated with multiple types.
In adults, RV-A is significantly more often detected in cases with acute cough/LRTI than RV-C, while RV-B infection is often found in asymptomatic patients.
•This study is the first to describe the safety and immunogenicity of a nanoporous microneedle array for SARS-CoV-2 vaccination with the mRNA-1273 vaccine in healthy participants as a booster ...vaccination.•Nanoporous microneedle array for SARS-CoV-2 vaccination with 20 µg mRNA-1273 was safe but failed to induce an anamnestic antibody response, which is probably due to the used vaccine loading technique resulting in a very low amount of loaded vaccine on the microneedle tips.•As microneedle patch immunisation is a promising vaccination technique; it is worth to evaluate this novel technology further to be better prepared for pandemics in the future.
Nanoporous microneedle arrays (npMNA) are being developed as skin patches for vaccine delivery. As alternative for needle-based immunisation, they may potentially result in higher vaccine acceptance, which is important for future mass vaccination campaigns to control outbreaks, such as COVID-19, and for public vaccination in general. In this study we investigated the safety and immunogenicity of needle-free intradermal delivery of a fractional third or fourth dose of mRNA-1273 vaccine by npMNA.
This study was an open-label, randomised-controlled, proof-of-concept study. Healthy adults were eligible if they had received a primary immunisation series against SARS-CoV-2 with two doses of mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) mRNA vaccine. A history of a COVID-19 infection or booster vaccination with mRNA-1273 or BNT162b2 was allowed if it occurred at least three months before inclusion. Participants were randomised in a 1:1 ratio to receive 20 µg mRNA-1273 vaccine, either through npMNA patch applied on the skin (ID-patch group), or through intramuscular (IM) injection (IM-control group). Primary outcomes were reactogenicity up to two weeks after vaccination, and fold-increase of SARS-CoV-2 spike S1-specific IgG antibodies 14 days post-vaccination.
In April 2022, 20 participants were enroled. The geometric mean concentration (GMC) did not increase in the ID-patch group after vaccination, in contrast to the IM-control group (GMC was 1,006 BAU/mL (95% CI 599–1,689), 3,855 (2,800–5,306), and 3,513 (2,554–4,833) at day 1, 15 and 29, respectively). In addition, SARS-CoV-2-specific T cell responses were lower after ID vaccination through npMNA.
Needle-free delivery of 20 µg mRNA-1273 vaccine by npMNA failed to induce antibody and T cell responses. As this is a potentially very useful vaccination method, it is important to determine which adjustments are needed to make this npMNA successful.
NCT05315362.
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.
Schistosomiasis is a parasitic disease affecting over 250 million people in the tropics. In non-endemic regions, imported
Schistosoma
infections are commonly diagnosed by serology, but based on ...antibody detection an active infection cannot be distinguished from a cured infection and it may take more than 8 weeks after exposure before seroconversion occurs. In endemic populations, excellent results have been described in diagnosing low-grade active
Schistosoma
infections by the detection of the adult worm-derived circulating anodic antigen (CAA) utilising robust lateral flow (LF) assays combined with up-converting phosphor (UCP) reporter technology. The purpose of this study is to explore the diagnostic value of the UCP-LF CAA assay in a non-endemic setting. CAA concentrations were determined in 111 serum samples originating from 81 serology-positive individuals. In nine individuals, serum could be collected before travel and an additional five provided samples before and after seroconversion occurred. Based on detectable CAA levels, an active infection was seen in 56/81 (69%) of the exposed individuals, while the 10 controls and the 9 sera collected before travel were tested negative for CAA. Positive CAA levels were observed starting 4 weeks after exposure and in four cases CAA was detected even before
Schistosoma
-specific antibodies became positive. Higher serum CAA levels were seen in migrants than in travellers and CAA concentrations dropped sharply when testing follow-up samples after treatment. This explorative study indicates the UCP-LF CAA serum assay to be a highly accurate test for detecting active low-grade
Schistosoma
infections in a non-endemic routine diagnostic setting.
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