Sensitivity of Detection and Variant Typing of SARS-CoV-2 in European Laboratories.

The molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key for clinical management and surveillance. Funded by the European Centre for Disease Prevention and Control, we conducted an external quality assessment (EQA) on the molecular detection and variant typing of SARS-CoV-2 that included 59 European laboratories in 34 countries. The EQA panel consisted of 12 lyophilized inactivated samples, 10 of which were SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Epsilon, Eta, parental B.1 strain) ranging from 2.5 to 290.0 copies/µL or pooled respiratory viruses (adenovirus, enterovirus, influenza virus A, respiratory syncytial virus, or human coronaviruses 229E and OC43). Of all participants, 72.9% identified the presence of SARS-CoV-2 RNA correctly. In samples containing 25.0 or more genome copies/µL, SARS-CoV-2 was detected by 98.3% of the participating laboratories. Laboratories applying commercial tests scored significantly better (P < 0.0001, Kruskal-Wallis test) than those using in-house assays. Both the molecular detection and the typing of the SARS-CoV-2 variants were associated with the RNA concentrations (P < 0.0001, Kruskal-Wallis test). On average, only 5 out of the 10 samples containing different SARS-CoV-2 variants at different concentrations were correctly typed. The identification of SARS-CoV-2 variants was significantly more successful among EQA participants who combined real-time reverse transcription polymerase chain reaction (RT-PCR)-based assays for mutation detection and high-throughput genomic sequencing than among those who used a single methodological approach (P = 0.0345, Kruskal-Wallis test). Our data highlight the high sensitivity of SARS-CoV-2 detection in expert laboratories as well as the importance of continuous assay development and the benefits of combining different methodologies for accurate SARS-CoV-2 variant typing.

Variable Sensitivity of SARS-CoV-2 Molecular Detection in European Expert Laboratories: External Quality Assessment, June and July 2020.

During the ongoing coronavirus disease 2019 (COVID-19) outbreak, robust detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key element for clinical management and to interrupt transmission chains. We organized an external quality assessment (EQA) of molecular detection of SARS-CoV-2 for European expert laboratories. An EQA panel composed of 12 samples, containing either SARS-CoV-2 at different concentrations to evaluate sensitivity or other respiratory viruses to evaluate specificity of SARS-CoV-2 testing, was distributed to 68 laboratories in 35 countries. Specificity samples included seasonal human coronaviruses hCoV-229E, hCoV-NL63, and hCoV-OC43, as well as Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and human influenza viruses A and B. Sensitivity results differed among laboratories, particularly for low-concentration SARS-CoV-2 samples. Results indicated that performance was mostly independent of the selection of specific extraction or PCR methods.

Towards a sensitive and accurate interpretation of molecular testing for SARS-CoV-2: a rapid review of 264 studies.

BackgroundSensitive molecular diagnostics and correct test interpretation are crucial for accurate COVID-19 diagnosis and thereby essential for good clinical practice. Furthermore, they are a key factor in outbreak control where active case finding in combination with isolation and contact tracing are crucial.AimWith the objective to inform the public health and laboratory responses to the pandemic, we reviewed current published knowledge on the kinetics of SARS-CoV-2 infection as assessed by RNA molecular detection in a wide range of clinical samples.MethodsWe performed an extensive search on studies published between 1 December 2019 and 15 May 2020, reporting on molecular detection and/or isolation of SARS-CoV-2 in any human laboratory specimen.ResultsWe compiled a dataset of 264 studies including 32,515 COVID-19 cases, and additionally aggregated data points (n = 2,777) from sampling of 217 adults with known infection timeline. We summarised data on SARS-CoV-2 detection in the respiratory and gastrointestinal tract, blood, oral fluid, tears, cerebrospinal fluid, peritoneal fluid, semen, vaginal fluid; where provided, we also summarised specific observations on SARS-CoV-2 detection in pregnancy, infancy, children, adolescents and immunocompromised individuals.ConclusionOptimal SARS-CoV-2 molecular testing relies on choosing the most appropriate sample type, collected with adequate sampling technique, and with the infection timeline in mind. We outlined knowledge gaps and directions for future well-documented systematic studies.

Serologic Detection of Middle East Respiratory Syndrome Coronavirus Functional Antibodies.

We developed and validated 2 species-independent protein-based assays to detect Middle East respiratory syndrome coronavirus functional antibodies that can block virus receptor-binding or sialic acid-attachment. Antibody levels measured in both assays correlated strongly with virus-neutralizing antibody titers, proving their use for serologic confirmatory diagnosis of Middle East respiratory syndrome.

Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Patients.

A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

Laboratory readiness and response for novel coronavirus (2019-nCoV) in expert laboratories in 30 EU/EEA countries, January 2020.

Timely detection of novel coronavirus (2019-nCoV) infection cases is crucial to interrupt the spread of this virus. We assessed the required expertise and capacity for molecular detection of 2019-nCoV in specialised laboratories in 30 European Union/European Economic Area (EU/EEA) countries. Thirty-eight laboratories in 24 EU/EEA countries had diagnostic tests available by 29 January 2020. A coverage of all EU/EEA countries was expected by mid-February. Availability of primers/probes, positive controls and personnel were main implementation barriers.

Whole-Blood Testing for Diagnosis of Acute Zika Virus Infections in Routine Diagnostic Setting.

We evaluated the benefit of whole blood versus plasma to detect acute Zika virus infections. Comparison of Zika virus quantitative reverse transcription PCR results in single timepoint whole blood-plasma pairs from 227 patients with suspected Zika virus infection resulted in confirmation of 8 additional patients with Zika virus infection.

Sensitive and Specific Detection of Low-Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavirus Infections.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein-based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43-positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.

Usutu virus infection in Dutch blood donors.

BACKGROUND: The screening of Dutch blood donations for West Nile virus (WNV) may be imminent, as WNV emerges in nearby countries and more donors travel to WNV-affected regions. Since 2016 the related, mosquito-borne Usutu virus (USUV) causes seasonal mortality in Dutch birds. To what extent will human USUV infections affect Dutch WNV donor screening? STUDY DESIGN AND METHODS: From April through September 2018, plasma samples from blood donations in blackbird-rich regions were stored. When increased bird mortality was reported in August, samples from July, August, and September were tested for USUV-RNA in pools of eight, using a home-brew combined WNV/USUV-PCR assay. Reactive pools were deconstructed. Original plasma units and samples of previous and follow-up donations of reactive donors were tested for USUV- and WNV-RNA, and for antibody responses. RESULTS: The number of USUV RNA-positive, WNV RNA-negative donations was 0 of 2688 donations in July, 6 of 4416 in August (1:736), and 1 of 4936 in September. The seven infected donors tested negative for USUV-RNA in preceding and follow-up donations. For 6 donors, seroconversion for USUV-antibodies was demonstrated. All index donations tested positive in a commonly used PCR-assay for WNV donor screening. Three exposed recipients did not show signs of infection. Screening a random subset of 1092 donations from September for USUV-IgG antibodies showed that 22 donors tested reactive; for three donors retrospective testing identified an USUV PCR-positive pre-seroconversion donation. CONCLUSION: Seasonal USUV infection in Dutch blood donors is common. Cross-reactivity in molecular assays for WNV-screening occurs, but can be resolved using USUV- and WNV-specific PCR-primers and sequencing of viral RNA.

Need for additional capacity and improved capability for molecular detection of yellow fever virus in European Expert Laboratories: External Quality Assessment, March 2018.

An external quality assessment of yellow fever virus (YFV) molecular detection in European laboratories was organised in rapid response to an increase in human cases in Brazil in 2018 with risk of import to Europe. Detection of YFV was assessed among 32 laboratories in 23/31 European Union (EU) and European Economic Area (EEA) countries and two laboratories in one non-EU/EEA country. Adequate capabilities were lacking in 10/23 countries; five did not participate as they lacked implemented assays.

Risk Factors for Primary Middle East Respiratory Syndrome Coronavirus Infection in Camel Workers in Qatar During 2013-2014: A Case-Control Study.

The transmission routes and risk factors for zoonotic Middle East respiratory syndrome coronavirus (MERS-CoV) infections are still unknown. We used the World Health Organization questionnaire for MERS-CoV case-control studies to assess risk factors for human MERS-CoV seropositivity at a farm complex in Qatar. Nine camel workers with MERS-CoV antibodies and 43 workers without antibodies were included. Some camel-related activities may pose a higher risk of MERS-CoV infection, as may cross-border movements of camels, poor hand hygiene, and overnight hospital stays with respiratory complaints. The risk factors identified in this study can be used to develop infection prevention and control measures for human MERS-CoV infections.

Ebola Virus Inactivation by Detergents Is Annulled in Serum.

Background: Treatment of blood samples from hemorrhagic fever virus (HFV)-infected patients with 0.1% detergents has been recommended for virus inactivation and subsequent safe laboratory testing. However, data on virus inactivation by this procedure are lacking. Here we show the effect of this procedure on diagnostic test results and infectious Ebola virus (EBOV) titers. Methods: Serum and whole-blood samples were treated with 0.1% or 1% sodium dodecyl sulfate (SDS) or 0.1% Triton X-100 and assayed for clinical chemistry and malaria antigen detection. Infectious EBOV titers were determined in SDS-treated plasma and whole blood from EBOV-infected nonhuman primates (NHPs). Infectious titers of EBOV or herpes simplex virus type 1 (HSV-1) in detergents-treated cell culture medium containing various serum concentrations were determined. Results: Laboratory test results were not affected by 0.1% detergent treatment of blood samples, in contrast with 1% SDS treatment. However, 0.1% detergent treatment did not inactivate EBOV in blood samples from infected NHPs. Experiments with cell culture medium showed that virus inactivation by detergents is annulled at physiological serum concentrations. Conclusions: Treatment of blood samples with 0.1% SDS or Triton X-100 does not inactivate EBOV. Inactivation protocols for HFV should be validated with serum and whole blood.

Variegated Squirrel Bornavirus 1 in Squirrels, Germany and the Netherlands.

We screened squirrels in Germany and the Netherlands for the novel zoonotic variegated squirrel bornavirus 1 (VSBV-1). The detection of VSBV-1 in 11 squirrels indicates a considerable risk for transmission to humans handling those animals. Therefore, squirrels in contact with humans should routinely be tested for VSBV-1.

Occupational Exposure to Dromedaries and Risk for MERS-CoV Infection, Qatar, 2013-2014.

We determined the presence of neutralizing antibodies to Middle East respiratory syndrome coronavirus in persons in Qatar with and without dromedary contact. Antibodies were only detected in those with contact, suggesting dromedary exposure as a risk factor for infection. Findings also showed evidence for substantial underestimation of the infection in populations at risk in Qatar.

Geographic distribution of MERS coronavirus among dromedary camels, Africa.

We found serologic evidence for the circulation of Middle East respiratory syndrome coronavirus among dromedary camels in Nigeria, Tunisia, and Ethiopia. Circulation of the virus among dromedaries across broad areas of Africa may indicate that this disease is currently underdiagnosed in humans outside the Arabian Peninsula.

Antibodies against MERS coronavirus in dromedary camels, United Arab Emirates, 2003 and 2013.

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most (389/651, 59.8%) serum samples had MERS-CoV-neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases.

Isolation of MERS coronavirus from a dromedary camel, Qatar, 2014.

We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels.

Ixodes ricinus as potential vector for Usutu virus.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands.

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.