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 α2,3- and α2,6-sialic acid. The model presented here could facilitate the development of a new generation of H2N2 influenza vaccines. IMPORTANCE In 1957 the world was subjected to a pandemic caused by an influenza A virus of the subtype H2N2. Although the virus disappeared in 1968, H2 viruses continue to circulate in avian reservoirs. It is therefore possible that the H2N2 influenza virus will be reintroduced into the human population, which can lead to another pandemic. The impact of a new H2N2 influenza pandemic can be mitigated by vaccination. However, these vaccines first need to be developed and tested in animal models. In preparation for this, we expanded the ferret model to mimic the different facets of human H2N2 influenza infection and disease. This model can be used for the development and evaluation of new H2N2 influenza vaccines.

SARS-CoV-2 RNA and antibody dynamics in a Dutch household study with dense sampling frame.

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.

Multi-center evaluation of Cepheid Xpert® Xpress SARS-CoV-2/Flu/RSV molecular point-of-care test.

Background: SARS-CoV-2 is taking a huge toll on society while influenza and RSV detection are also becoming more important. These viruses pose a high burden on health care. Rapid and accurate diagnostics for these pathogens are important for swift triage in the hospital. Fast molecular point of care test (mPOCT) assays for these pathogens can prove an alternative. Here a multi-center evaluation of the Xpert® Xpress SARS-CoV-2/Flu/RSV assay is reported. Study design: The Xpert® Xpress SARS-CoV-2/Flu/RSV assay was compared to three reference assays at three Dutch medical microbiology laboratories. An external quality assessment panel consisting of 16 specimens containing SARS-CoV-2, influenza viruses, RSV or human seasonal coronaviruses, or a combination thereof were used. Clinical specimens containing SARS-CoV-2 (n = 57), influenza viruses (n = 21) or RSV (n = 12), at a wide range of relevant concentrations were used. One laboratory also tested zoonotic avian and swine influenza viruses, and eight relevant SARS-CoV-2 variants. Results: The Xpert® Xpress SARS-CoV-2/Flu/RSV assay showed equal performance compared to the reference assays. All SARS-CoV-2 variants of interest and variants of concern were accurately detected. Human seasonal coronaviruses were not detected. All four circulating seasonal influenza virus subtypes/lineages and both RSV types were accurately detected as well as a set of recent zoonotic avian and swine influenza viruses. The clinical specimens showed 98.2% concordance using this assay. Conclusion: The Xpert® Xpress SARS-CoV-2/Flu/RSV assay is a good alternative for accurate detection for SARS-CoV-2, influenza type A virus, influenza type B virus and RSV types A and B detection in a short timeframe.

Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic.

BACKGROUND: With the outbreak of SARS-CoV-2, rapid diagnostics are paramount to contain the current pandemic. The routinely used realtime RT-PCR is sensitive, specific and able to process large batches of samples. However, turnaround time is long and in cases where fast obtained results are critical, molecular point of care tests (POCT) can be an alternative. Here we report on a multicenter evaluation of the Cepheid Xpert Xpress SARS-CoV-2 point-of-care test. STUDY DESIGN: The Xpert Xpress SARS-CoV-2 assay was evaluated against the routine in-house real-time RT-PCR assays in three medical microbiology laboratories in The Netherlands. A sensitivity and specificity panel was tested consisting of a dilution series of SARS-CoV-2 and ten samples containing SARS-CoV-2 and a range of other seasonal respiratory viruses. Additionally, 58 samples of patients positive for SARS-CoV-2 with different viral loads and 30 tested negative samples in all three Dutch laboratories using an in-house RT-PCR, were evaluated using Cepheids Xpert Xpress SARS-CoV-2 cartridges. RESULTS: Xpert Xpress SARS-CoV-2 point of care test showed equal performance compared to routine in-house testing with a limit of detection (LOD) of 8.26 copies/mL. Other seasonal respiratory viruses were not detected. In clinical samples Xpert Xpress SARS-CoV-2 reaches an agreement of 100 % compared to all in-house RT-PCRs CONCLUSION: Cepheids GeneXpert Xpert Xpress SARS-CoV-2 is a valuable addition for laboratories in situations where rapid and accurate diagnostics are of the essence.

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.

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

BACKGROUND: 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. AIM: We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. METHODS: 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. RESULTS: 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. CONCLUSION: The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

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.

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.