Comparison of seven commercial RT-PCR diagnostic kits for COVID-19

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

Varying Viral Replication and Disease Profiles of H2N2 Influenza in Ferrets Is Associated with Virus Isolate and Inoculation Route

H2N2 influenza virus, the causative agent of the 1957 “Asian flu” pandemic, has disappeared from circulation. However, H2-influenza viruses are still circulating in avian reservoirs. Combined with the waning of H2N2-specific immunity in the human population, there is a risk of reintroduction of H2N2 influenza virus. Vaccines could help in preventing a future pandemic, but to assess their efficacy animal models are required. We therefore set out to expand the ferret model for H2N2 influenza disease by infecting ferrets intranasally or intratracheally with four different H2N2 viruses to investigate their influence on the severity of disease. The H2N2 viruses were collected either during the pandemic or near the end of H2N2 circulation and covered both clade I and clade II viruses. Infection of ferrets with the different viruses showed that viral replication, disease, and pathology differed markedly between virus isolates and infection routes. Intranasal inoculation induced a severe to mild rhinitis, depending on the virus isolate, and did not lead to lung infection or pathology. When administered intratracheally, isolates that successfully replicated in the lower respiratory tract (LRT) induced a nonlethal disease that resembles that of a moderate pneumonia in humans. Differences in viral replication and disease between viruses could be associated with their binding preference for a2,3- and a2,6-sialic acid. The model presented here could facilitate the development of a new generation of H2N2 influenza vaccines

Case of seasonal reassortant A(H1N2) influenza virus infection, the Netherlands, March 2018.

A seasonal reassortant A(H1N2) influenza virus harbouring genome segments from seasonal influenza viruses A(H1N1)pdm09 (HA and NS) and A(H3N2) (PB2, PB1, PA, NP, NA and M) was identified in March 2018 in a 19-months-old patient with influenza-like illness (ILI) who presented to a general practitioner participating in the routine sentinel surveillance of ILI in the Netherlands. The patient recovered fully. Further epidemiological and virological investigation did not reveal additional cases

Respiratory syncytial, parainfluenza and influenza virus infection in young children with acute lower respiratory infection in rural Gambia.

Respiratory viral infections contribute significantly to morbidity and mortality worldwide, but representative data from sub-Saharan Africa are needed to inform vaccination strategies. We conducted population-based surveillance in rural Gambia using standardized criteria to identify and investigate children with acute lower respiratory infection (ALRI). Naso- and oropharyngeal swabs were collected. Each month from February through December 2015, specimens from 50 children aged 2-23 months were randomly selected to test for respiratory syncytial (RSV), parainfluenza (PIV) and influenza viruses. The expected number of viral-associated ALRI cases in the population was estimated using statistical simulation that accounted for the sampling design. RSV G and F proteins and influenza hemagglutinin genes were sequenced. 2385 children with ALRI were enrolled, 519 were randomly selected for viral testing. One or more viruses were detected in 303/519 children (58.4%). RSV-A was detected in 237 and RSV-B in seven. The expected incidence of ALRI associated with RSV, PIV or influenza was 140 cases (95% CI, 131-149) per 1000 person-years; RSV incidence was 112 cases (95% CI, 102-122) per 1000 person-years. Multiple strains of RSV and influenza circulated during the year. RSV circulated throughout most of the year and was associated with eight times the number of ALRI cases compared to PIV or IV. Gambian RSV viruses were closely related to viruses detected in other continents. An effective RSV vaccination strategy could have a major impact on the burden of ALRI in this setting

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

BackgroundThe 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.AimWe aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.MethodsHere 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.ResultsThe 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.ConclusionThe present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments

Case of seasonal reassortant a(H1N2) influenza virus infection, the Netherlands, March 2018

A seasonal reassortant A(H1N2) influenza virus harbouring genome segments from seasonal influenza viruses A(H1N1)pdm09 (HA and NS) and A(H3N2) (PB2, PB1, PA, NP, NA and M) was identified in March 2018 in a 19-months-old patient with influenza-like illness (ILI) who presented to a general practitioner participating in the routine sentinel surveillance of ILI in the Netherlands. The patient recovered fully. Further epidemiological and virological investigation did not reveal additional cases

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.</p

Global maps of soil temperature

Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km² resolution for 0–5 and 5–15 cm soil depth. These maps were created by calculating the difference (i.e., offset) between in-situ soil temperature measurements, based on time series from over 1200 1-km² pixels (summarized from 8500 unique temperature sensors) across all the world’s major terrestrial biomes, and coarse-grained air temperature estimates from ERA5-Land (an atmospheric reanalysis by the European Centre for Medium-Range Weather Forecasts). We show that mean annual soil temperature differs markedly from the corresponding gridded air temperature, by up to 10°C (mean = 3.0 ± 2.1°C), with substantial variation across biomes and seasons. Over the year, soils in cold and/or dry biomes are substantially warmer (+3.6 ± 2.3°C) than gridded air temperature, whereas soils in warm and humid environments are on average slightly cooler (-0.7 ± 2.3°C). The observed substantial and biome-specific offsets emphasize that the projected impacts of climate and climate change on near-surface biodiversity and ecosystem functioning are inaccurately assessed when air rather than soil temperature is used, especially in cold environments. The global soil-related bioclimatic variables provided here are an important step forward for any application in ecology and related disciplines. Nevertheless, we highlight the need to fill remaining geographic gaps by collecting more in-situ measurements of microclimate conditions to further enhance the spatiotemporal resolution of global soil temperature products for ecological applications

Micromechanical Properties of Injection-Molded Starch–Wood Particle Composites

The micromechanical properties of injection molded starch–wood particle composites were investigated as a function of particle content and humidity conditions. The composite materials were characterized by scanning electron microscopy and X-ray diffraction methods. The microhardness of the composites was shown to increase notably with the concentration of the wood particles. In addition,creep behavior under the indenter and temperature dependence were evaluated in terms of the independent contribution of the starch matrix and the wood microparticles to the hardness value. The influence of drying time on the density and weight uptake of the injection-molded composites was highlighted. The results revealed the role of the mechanism of water evaporation, showing that the dependence of water uptake and temperature was greater for the starch–wood composites than for the pure starch sample. Experiments performed during the drying process at 70°C indicated that the wood in the starch composites did not prevent water loss from the samples.Peer reviewe