The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is ...posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.
The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).
We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95 % limit of detection (LOD95). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n = 13) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n = 6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.
PCR efficiency was ≥96 % for all assays and the estimated LOD95 varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.
We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.
Sapoviruses (SaVs) belong to the Caliciviridae family and can cause gastroenteritis in humans and swine. Despite extensive testing, human sapoviruses have been found only in sporadic cases and in one ...mixed outbreak in children between 1994 and 2007 in the Netherlands. Here we describe a change in sapovirus epidemiology in the Netherlands resulting in sapovirus outbreaks and infections in adults. From November 2007 to January 2009, 478 outbreaks of acute gastroenteritis were reported to the National Institute for Public Health and the Environment in the Netherlands as a part of ongoing surveillance. Sapoviruses were found to be the most likely cause of 19 outbreaks (4%). During the same 2-year period, sapovirus infections were reported in Sweden, Slovenia, and Hungary. In the Netherlands, further characterization of outbreak strains showed that 12 (63%) sapovirus outbreaks were caused by genotype I.2 viruses. Most patients were adults older than 60 years (range, 1 to 100 years). Phylogenetic analysis using all presently available SaV sequences showed high homology between genotype I.2 strains detected in different geographical regions (Sweden, Slovenia, Taiwan, Japan, and Russia) since 2007. These first reported outbreaks of sapovirus infections in adults in the Netherlands were remarkable. Detection of identical genotypes in many samples might suggest that these viruses have the same origin, and since the infection is spreading fast, the prevalence of sapovirus infection may be increasing. The incidence of sapovirus infections in these countries suggests that a substantial part of Europe is affected by this virus.
This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in 242 household members of different ages and with different symptom severity after SARS-CoV-2 exposure early in the ...pandemic (March-April 2020). Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.
The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the ...outbreak is more widespread than initially thought, and international spread through travellers does already occur.
We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.
Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.
The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project.
The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
Norovirus is the primary cause of viral gastroenteritis (GE). To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of complete genomes. To ...investigate the potential of shotgun metagenomic sequencing on the Illumina platform for whole-genome sequencing, 71 reverse transcriptase quantitative PCR (RT-qPCR) norovirus positive-feces (threshold cycle
, <30) samples from norovirus surveillance within The Netherlands were subjected to metagenomic sequencing. Data were analyzed through an in-house next-generation sequencing (NGS) analysis workflow. Additionally, we assessed the potential of metagenomic sequencing for the surveillance of off-target viruses that are of importance for public health, e.g., sapovirus, rotavirus A, enterovirus, parechovirus, aichivirus, adenovirus, and bocaparvovirus. A total of 60 complete and 10 partial norovirus genomes were generated, representing 7 genogroup I capsid genotypes and 12 genogroup II capsid genotypes. In addition to the norovirus genomes, the metagenomic approach yielded partial or complete genomes of other viruses for 39% of samples from children and 6.7% of samples from adults, including adenovirus 41 (
= 1); aichivirus 1 (
= 1); coxsackievirus A2 (
= 2), A4 (
= 2), A5 (
= 1), and A16 (
= 1); bocaparvovirus 1 (
= 1) and 3 (
= 1); human parechovirus 1 (
= 2) and 3 (
= 1); Rotavirus A (
= 1); and a sapovirus GI.7 (
= 1). The sapovirus GI.7 was initially not detected through RT-qPCR and warranted an update of the primer and probe set. Metagenomic sequencing on the Illumina platform robustly determines complete norovirus genomes and may be used to broaden gastroenteritis surveillance by capturing off-target enteric viruses.
Viral gastroenteritis results in significant morbidity and mortality in vulnerable individuals and is primarily caused by norovirus. To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of full genomes. Using surveillance samples sent to the Dutch National Institute for Public Health and the Environment (RIVM), we compared metagenomics against conventional techniques, such as RT-qPCR and Sanger-sequencing, with norovirus as the target pathogen. We determined that metagenomics is a robust method to generate complete norovirus genomes, in parallel to many off-target pathogenic enteric virus genomes, thereby broadening our surveillance efforts. Moreover, we detected a sapovirus that was not detected by our validated gastroenteritis RT-qPCR panel, which exemplifies the strength of metagenomics. Our study shows that metagenomics can be used for public health gastroenteritis surveillance, the generation of reference-sets for molecular epidemiology, and how it compares to current surveillance strategies.
Acute gastroenteritis is one of the most common diseases worldwide. In developed countries, viruses, particularly noroviruses, are recognized as the leading cause. In The Netherlands, the ...surveillance of gastroenteritis outbreaks with suspected viral etiologies (as determined by Kaplan criteria) was established by the National Institute for Public Health and the Environment in 1994. This paper presents an overview of viral gastroenteritis outbreaks reported from 1994 through 2005. A minimum epidemiological data set consisting of the associated setting(s), the probable transmission mode, the date of the first illness and the date of sampling, the number of persons affected, and the number of hospitalizations was requested for each reported outbreak. Stool samples were tested for the presence of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, and Aichi virus by electron microscopy, enzyme-linked immunosorbent assay, and/or reverse transcription-PCR. A total of 6,707 stool samples from 941 gastroenteritis outbreaks were investigated. Noroviruses were detected as the causative agent in 735 (78.1%) of the outbreaks, and rotaviruses, adenoviruses, and astroviruses were found to be responsible for 46 (4.9%), 9 (1.0%), and 5 (0.5%) outbreaks, respectively. Among the gastroenteritis outbreaks in which a mode of transmission was identified, most outbreaks (38.1%) were associated with person-to-person transmission, and the majority (54.9%) of the outbreaks investigated were reported by residential institutions. Since 2002, the total number of outbreaks reported and the number of unexplained outbreaks have increased. Furthermore, the number of rotavirus-associated outbreaks has increased, especially in nursing homes. Despite thorough testing, 115 (12.2%) outbreaks suspected of having viral etiologies remain unexplained. Increases in numbers of reported outbreaks may indicate undefined changes in the criteria for reporting or the emergence of new pathogens.
Human metapneumovirus (HMPV) is one of the leading causes of respiratory illness (RI), primarily in infants. Worldwide, two genetic lineages (A and B) of HMPV are circulating that are antigenically ...distinct and can each be further divided into genetic sublineages. Surveillance combined with large-scale whole-genome sequencing studies of HMPV are scarce but would help to identify viral evolutionary dynamics. Here, we analyzed 130 whole HMPV genome sequences obtained from samples collected from individuals hospitalized with RI and partial fusion (
= 144) and attachment (
= 123) protein gene sequences obtained from samples collected from patients with RI visiting general practitioners between 2005 and 2021 in the Netherlands. Phylogenetic analyses demonstrated that HMPV continued to group in the four sublineages described in 2004 (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected in the Netherlands after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between samples obtained from patients with RI being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. In the past, different names for the newly energing lineages have been proposed, demonstrating the need for a consistent naming convention. Here, criteria are proposed for the designation of new genetic lineages to aid in moving toward a systematic HMPV classification.
Human metapneumovirus (HMPV) is one of the major causative agents of human respiratory tract infections. Monitoring of virus evolution could aid toward the development of new antiviral treatments or vaccine designs. Here, we studied HMPV evolution between 2005 and 2021, with viruses obtained from samples collected from hospitalized individuals and patients with respiratory infections consulting general practitioners. Phylogenetic analyses demonstrated that HMPV continued to group in the four previously described sublineages (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between patients being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. These data were used to propose criteria for the designation of new genetic lineages to aid toward a systematic HMPV classification.
The extent to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) break through infection- or vaccine-induced immunity is not well understood. We analyzed ...28,578 sequenced SARS-CoV-2 samples from individuals with known immune status obtained through national community testing in the Netherlands from March to August 2021. We found evidence of an increased risk of infection by the Beta (B.1.351), Gamma (P.1), or Delta (B.1.617.2) variants compared with the Alpha (B.1.1.7) variant after vaccination. No clear differences were found between vaccines. However, the effect was larger in the first 14 to 59 days after complete vaccination compared with ≥60 days. In contrast to vaccine-induced immunity, there was no increased risk for reinfection with Beta, Gamma, or Delta variants relative to the Alpha variant in individuals with infection-induced immunity.
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