Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics,... Show moreViruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics, sensitive and simultaneous detection of RNA and DNA viruses is needed. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. The sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. Subsequently, the protocol was retrospectively validated using 25 clinical respiratory samples. The developed protocol using Illumina NextSeq 500 sequencing showed high repeatability. Use of the National Center for Biotechnology Information's RefSeq database as opposed to the National Center for Biotechnology Information's nucleotide database led to enhanced specificity of classification of viral pathogens. A correlation was established between read counts and PCR cycle threshold value. Sensitivity of mNGS, compared with PCR, varied up to 83%, with specificity of 94%, dependent on the cutoff for defining positive mNGS results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow, were influenza C, KI polyomavirus, cytomegalovirus, and enterovirus. Sensitivity and analytical specificity of this mNGS protocol were comparable to PCR and higher when considering off-PCR target viral pathogens. One single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses. Show less
Ogando, N.S.; Dalebout, T.J.; Zevenhoven-Dobbe, J.C.; Limpens, R.W.A.L.; Meer, Y. van der; Caly, L.; ... ; Snijder, E.J. 2020
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... Show moreThe 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. lmmunofluorescence 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. Show less
Background: Diagnosing pneumonia can be challenging in general practice but is essential to distinguish from other respiratory tract infections because of treatment choice and outcome prediction.... Show moreBackground: Diagnosing pneumonia can be challenging in general practice but is essential to distinguish from other respiratory tract infections because of treatment choice and outcome prediction. We determined predictive signs, symptoms and biomarkers for the presence of pneumonia in patients with acute respiratory tract infection in primary care.Methods: From March 2012 until May 2016 we did a prospective observational cohort study in three radiology departments in the Leiden-The Hague area, The Netherlands. From adult patients we collected clinical characteristics and biomarkers, chest X ray results and outcome. To assess the predictive value of C-reactive protein (CRP), procalcitonin and midregional pro-adrenomedullin for pneumonia, univariate and multivariate binary logistic regression were used to determine risk factors and to develop a prediction model.Results: Two hundred forty-nine patients were included of whom 30 (12%) displayed a consolidation on chest X ray. Absence of runny nose and whether or not a patient felt ill were independent predictors for pneumonia. CRP predicts pneumonia better than the other biomarkers but adding CRP to the clinical model did not improve classification (- 4%); however, CRP helped guidance of the decision which patients should be given antibiotics.Conclusions: Adding CRP measurements to a clinical model in selected patients with an acute respiratory infection does not improve prediction of pneumonia, but does help in giving guidance on which patients to treat with antibiotics. Our findings put the use of biomarkers and chest X ray in diagnosing pneumonia and for treatment decisions into some perspective for general practitioners. Show less
Rijn, A.L. van; Boheemen, S. van; Sidorov, I.; Carbo, E.C.; Pappas, N.; Mei, H.; ... ; Vries, J.J.C. de 2019
IntroductionExacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an... Show moreIntroductionExacerbations 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.ObjectivesTo study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS.Study design88 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.ResultsBy 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.ConclusionsThe 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. Show less
Groeneveld, G.H.; Reyden, T.J. van der; Joosten, S.A.; Bootsma, H.J.; Cobbaert, C.M.; Vries, J.J.C. de; ... ; Dissel, J.T. van 2019
AbstractBackground: Respiratory infections are considered a potential risk of adverse events in children undergoingsurgery. Rhinovirus is a common cause of respiratory infections and congenital... Show moreAbstractBackground: Respiratory infections are considered a potential risk of adverse events in children undergoingsurgery. Rhinovirus is a common cause of respiratory infections and congenital heart disease is a risk factor forsevere rhinovirus infection. However, we do not know what the impact of, clinical or subclinical, rhinovirus infectionsis on postoperative course following congenital heart surgery in children.Based on our clinical experience, one case-controlled study, and a case reported in the literature, we hypothesizethat paediatric patients with per-operative rhinovirus positive Polymerase Chain Reaction testing have a longerpaediatric intensive care unit admission, compared to children who test negative.Methods/Design: This is a prospective single-center observational study in the Leiden University Medical Centerwith approximately 250 children (<12 years) undergoing elective cardiac surgery, for congenital heart disease.The parents/guardians of the children will be asked to fill out a questionnaire, to asses respiratory symptoms inthe last weeks, before the operation of their child. In the operating theatre, a nasopharyngeal swab will be collected.Clinical data will be collected daily during paediatric intensive care admission and paediatric intensive care unit andhospital length of stay will be recorded. If children are still intubated at day 4, a second nasopharyngeal swab andresidual blood will be collected. The samples will be tested for rhinovirus with polymerase chain reaction. Primaryoutcome is the paediatric intensive care unit length of stay in per-operative rhinovirus-positive compared torhinovirus-negative patients.Discussion: This is the first study to screen children for rhinovirus before undergoing cardiac surgery and tostudy the effects on paediatric intensive care unit length of stay. Furthermore, we aim to identify children at risk forprolonged paediatric intensive care admission after cardiac surgery. Show less
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... Show moreOur 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. Show less
Berg-van de Glind, G.J.D. van den; Vries, J.J.C. de; Wolthers, K.C.; Wiggers-de Bruine, F.T.; Peeters-Scholte, C.M.P.C.D.; Hendef, M. van den; Wezel-Meijler, G. van 2012
Cytomegalovirus (CMV) infection is the most common congenital viral infection worldwide. The symptom of congenital CMV infection encountered most frequently is sensorineural hearing loss, which... Show moreCytomegalovirus (CMV) infection is the most common congenital viral infection worldwide. The symptom of congenital CMV infection encountered most frequently is sensorineural hearing loss, which will affect approximately one out of __ve congenitally infected newborns. Because of the late-onset nature of the hearing loss, up to half of the children with congenital CMV-related hearing loss may not be detected in the newborn hearing screening. This thesis addresses several aspects of congenital CMV infection in general and more specifically in the Netherlands, in order to determine the necessity and feasibility of newborn screening for congenital CMV. Show less
Congenital cytomegalovirus (CMV) infection is the most common congenital viral infection worldwide. The sequela encountered most frequently is hearing impairment, affecting approximately one out of... Show moreCongenital cytomegalovirus (CMV) infection is the most common congenital viral infection worldwide. The sequela encountered most frequently is hearing impairment, affecting approximately one out of five infants congenitally infected. Data on the birth prevalence and risk factors of congenital CMV infection in the Netherlands are scarce. The aim of this study was to determine the birth prevalence of congenital CMV in the Netherlands. A sample of 6,500 dried blood spots (DBS) from infants born in the Netherlands was tested anonymously for CMV DNA. The sample was stratified by the number of live births in different regions of the Netherlands of the year 2007. Additionally, on a regional level, risk factors for congenital CMV were analyzed. The birth prevalence of congenital CMV in the Netherlands was 0.54% (35/6,433, 95%Cl 0.36-0.72). Congenital CMV infection was significantly higher in regions with more than 15% young children (0 5 years) compared with regions with a lower proportion of young children (OR 5.9, 95%Cl 1.4-25.2). Congenital CMV infection was significantly higher in regions with more than 30% immigrants compared with regions with a lower proportion of immigrants (OR 2.2, 95%Cl 1.1-4.6). This association was strongest for regions with more than 30% non-Western immigrants (OR 3.3, 95%Cl 1.5-7.5). Based on the knowledge of the natural history of congenital CMV infection, approximately 1,000 children are born with congenital CMV infection in the Netherlands annually, of whom eventually approximately 180 children (0.1% of all newborns) will be affected by long term sequelae, with hearing loss being the symptom encountered most frequently. J. Med. Virol. 83: 1777-1782, 2011. (C) 2011 Wiley-Liss, Inc. Show less
