Coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness.

Introduction: The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness.

Aim: The aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness.

Rhinovirus viremia in adult patients with high viral load in bronchoalveolar lavages.

Background: In children, rhinovirus viremia has been associated with higher nasopharyngeal loads and increase in severity of clinical signs and symptoms.

Objectives: This study aims to detect rhinovirus viremia in adult patients and to establish potential correlations with the clinical course.

Study Design: Adult patients with rhinovirus strongly positive bronchoalveolar lavages (BAL, quantitation cycle, Cq values <25) detected between 2008 and 2014 were studied retrospectively.

Phylogenetic evidence for underreporting of male-to-male sex among human immunodeficiency virus-infected donors in the Netherlands and Flanders.

Background: Separate transmission networks for human immunodeficiency virus (HIV) coexist. Molecular typing of viral genomes can provide insight in HIV transmission routes in donors for whom risk behavior-based donor selection failed.

Study Design And Methods: This study includes all HIV-infected Dutch and Flemish donors in the period 2005 to 2014 (n = 55).

VIRO-TypeNed, systematic molecular surveillance of enteroviruses in the Netherlands between 2010 and 2014.

VIRO-TypeNed is a collaborative molecular surveillance platform facilitated through a web-based database. Genetic data in combination with epidemiological, clinical and patient data are shared between clinical and public health laboratories, as part of the surveillance underpinning poliovirus eradication. We analysed the combination of data submitted from 2010 to 2014 to understand circulation patterns of non-polio enteroviruses (NPEV) of public health relevance.

Increase in ECHOvirus 6 infections associated with neurological symptoms in the Netherlands, June to August 2016.

The Dutch virus-typing network VIRO-TypeNed reported an increase in ECHOvirus 6 (E-6) infections with neurological symptoms in the Netherlands between June and August 2016. Of the 31 cases detected from January through August 2016, 15 presented with neurological symptoms. Ten of 15 neurological cases were detected in the same province and the identified viruses were genetically related.

Typing Pseudomonas aeruginosa Isolates with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry.

The introduction of standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms in the medical microbiological practice has revolutionized the way microbial species identification is performed on a daily basis. To a large extent, this is due to the ease of operation. Acquired spectra are compared to profiles obtained from cultured colonies present in a reference spectra database.

Reactivation of Human Herpes Virus-6 After Pediatric Stem Cell Transplantation: Risk Factors, Onset, Clinical Symptoms and Association With Severity of Acute Graft-Versus-Host Disease.

Background And Methods: To study clinical symptoms, timing and consequences of human herpesvirus-6 (HHV-6) reactivation after pediatric allogeneic stem cell transplantation (SCT), HHV-6 was investigated by plasma polymerase chain reaction in a cohort of 106 pediatric SCT recipients.

Results: HHV-6 viremia was detected post-SCT in 48% of the patients with a median time of onset at 20 days after SCT. In week 3 and 4 post-SCT, HHV-6 is the most common infectious agent detected.

Clinical evaluation of viral acute respiratory tract infections in children presenting to the emergency department of a tertiary referral hospital in the Netherlands.

Background: The relative incidence and clinical impact of individual respiratory viruses remains unclear among children presenting to the hospital emergency department with acute respiratory tract infection (ARTI).

Methods: During two winter periods, respiratory virus real-time multiplex PCR results were evaluated from children (< 18 years) presenting to the emergency department of a tertiary referral hospital with ARTI that had been sampled within 48 hours of hospital presentation. In an attempt to identify virus-specific distinguishing clinical features, single virus infections were correlated with presenting signs and symptoms, clinical findings and outcomes using multivariate logistic regression.

Prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness.

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.

Mass spectrometry-based comparative sequence analysis for the genetic monitoring of influenza A(H1N1)pdm09 virus.

The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed.

Post-travel screening of asymptomatic long-term travelers to the tropics for intestinal parasites using molecular diagnostics.

The incidence of asymptomatic travel-related parasitic infection is uncertain. Previous studies did not distinguish new incident infections, from past infections. Regardless of symptoms, we performed multiplex real-time polymerase chain reaction on pre- and post-travel stool samples of Dutch long-term travelers to the (sub)tropics.

Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Escherichia coli.

Resistance to ciprofloxacin in Escherichia coli is increasing parallel to increased use of fluoroquinolones both in The Netherlands and in other European countries. The objective was to investigate the contribution of active efflux and expression of outer membrane proteins (OMPs) in a collection of clinical E. coli isolates collected at a clinical microbiology department in a Dutch hospital.

Performance of the xTAG® gastrointestinal pathogen panel, a multiplex molecular assay for simultaneous detection of bacterial, viral, and parasitic causes of infectious gastroenteritis.

The xTAG(®) Gastrointestinal Pathogen Panel (GPP) is a multiplexed molecular test for 15 gastrointestinal pathogens. The sensitivity and specificity of this test were assessed in 901 stool specimens collected from pediatric and adult patients at four clinical sites. A combination of conventional and molecular methods was used as comparator.

September through October 2010 multi-centre study in the Netherlands examining laboratory ability to detect enterovirus 68, an emerging respiratory pathogen.

During September and October 2010, the Dutch Public Health Institute detected an enterovirus (EV) 68 (EV68) epidemic in the Netherlands through general practitioner-based surveillance of acute respiratory infections. EV68 shares phenotypic and genotypic properties with human rhinovirus (HRV). Despite increased EV and HRV detections, Dutch clinical laboratories did not identify EV68.

RT-PCR detection of respiratory pathogens in newborn children admitted to a neonatal medium care unit.

Background: This study aimed to determine the prevalence of respiratory pathogens among newborns admitted to a neonatal medium care unit (NMCU) and to identify clinical predictors.

Methods: A 1-y observational study was performed of neonates admitted to an NMCU in Amsterdam, The Netherlands. Nasopharyngeal samples were collected for the detection of respiratory viruses and bacteria by real-time PCR (RT-PCR).

Persistence and antiviral resistance of varicella zoster virus in hematological patients.

Background: Varicella zoster virus (VZV) infections are a relevant cause of morbidity and mortality in hematological patients and especially in hematopoietic stem cell transplant (HSCT) recipients. The present study aimed to investigate the prevalence and clinical significance of viral persistence and antiviral resistance by systematically analyzing all episodes of VZV diagnosed in our laboratory in pediatric and adult hematological patients between 2007 and 2010.

Methods: Patient charts were reviewed to document patient and disease characteristics.

No novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide CODEHOP-based primers.

Novel viruses might be responsible for numerous disease cases with unknown etiology. In this study, we screened 1800 nasopharyngeal samples from adult outpatients with respiratory disease symptoms and healthy individuals. We employed a reverse transcription (RT)-PCR assay and CODEHOP-based primers (CT12-mCODEHOP) previously developed to recognize known and unknown corona- and toroviruses.

Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR.

Background: Susceptibility testing of herpes simplex virus type 1 (HSV-1) is traditionally performed by a plaque reduction assay (PRA), but this is labor intensive, time consuming and has a manual read out.

Objectives: The goal of this study was to develop an internally controlled real time PCR-based phenotypical susceptibility test for HSV-1 that is suitable for use in a clinical diagnostic setting.

Study Design: A DNA reduction assay (DRA) was developed and validated on a test panel of 26 well-characterized isolates of varying susceptibility to aciclovir or foscarnet, including low-level resistant isolates.

Extraction of DNA from dried blood in the diagnosis of congenital CMV infection.

Viral DNA detection in dried blood spotted on filter paper, dried blood spots (DBS), is valuable in the diagnosis of viral infections, with at the moment congenital cytomegalovirus (CMV) being the most common application. CMV detection in clinical samples taken within the first 2-3 weeks after birth differentiates congenital CMV infection from the in general harmless postnatal acquired cytomegalovirus infection. DBS render the possibility to diagnose congenital CMV infection retrospectively, e.

Evaluation of several biochemical and molecular techniques for identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and their detection in respiratory samples.

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia.

Failure of pre-emptive treatment of cytomegalovirus infections and antiviral resistance in stem cell transplant recipients.

Background: Treatment of cytomegalovirus (CMV) infections after stem cell transplantation (SCT) does not always lead to a rapid viral response. The causes of treatment failure may be either viral resistance or immunological failure to control viral replication. This study investigated the response to pre-emptive treatment in CMV infections in order to define risk factors for treatment failure, including the role of antiviral resistance.

Real-time PCR versus viral culture on urine as a gold standard in the diagnosis of congenital cytomegalovirus infection.

Background: Cytomegalovirus (CMV) infection is the most common cause of congenital infection. Whereas CMV PCR has replaced viral culture and antigen detection in immunocompromised patients because of higher sensitivity, viral culture of neonatal urine is still referred to as the gold standard in the diagnosis of congenital CMV infection.

Objective: To compare real-time CMV PCR with shell vial culture on urine in the diagnosis of congenital CMV, in a multicenter design.

Congenital cytomegalovirus infection in the Netherlands: birth prevalence and risk factors.

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 five infants congenitally infected. Data on the birth prevalence and risk factors of congenital CMV infection in the Netherlands are scarce.

Etiology of suspected pneumonia in adults admitted to a high-dependency unit in Blantyre, Malawi.

The microbiologic etiology of severe pneumonia in hospitalized patients is rarely known in sub-Saharan Africa. Through a comprehensive diagnostic work-up, we aimed to identify the causative agent in severely ill patients with a clinical picture of pneumonia admitted to a high-dependency unit. A final diagnosis was made and categorized as confirmed or probable by using predefined criteria.

High titers of pre-existing adenovirus serotype-specific neutralizing antibodies in the host predict viral reactivation after allogeneic stem cell transplantation in children.

Background: Human adenovirus (HAdV) infections are frequent in children after allogeneic stem cell transplantation (SCT) and may become fatal. Whether these infections occur through reactivation of endogenous virus or transmission via the graft remains a matter of debate.

Methods: In a cohort of 24 pediatric patients who received SCT, infections with 1 or more of 5 serotypes of HAdV (1, 2, 5, 6, and 31) were detected by culture.