Variable detection of Omicron-BA.1 and -BA.2 by SARS-CoV-2 rapid antigen tests.

During 2022, the COVID-19 pandemic has been dominated by the variant of concern (VoC) Omicron (B.1.1.529) and its rapidly emerging subvariants, including Omicron-BA.1 and -BA.2. Rapid antigen tests (RATs) are part of national testing strategies to identify SARS-CoV-2 infections on site in a community setting or to support layman's diagnostics at home. We and others have recently demonstrated an impaired RAT detection of infections caused by Omicron-BA.1 compared to Delta. Here, we evaluated the performance of five SARS-CoV-2 RATs in a single-centre laboratory study examining a total of 140 SARS-CoV-2 PCR-positive respiratory swab samples, 70 Omicron-BA.1 and 70 Omicron-BA.2, as well as 52 SARS-CoV-2 PCR-negative swabs collected from March 8th until April 10th, 2022. One test did not meet minimal criteria for specificity. In an assessment of the analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 4.2 × 104 to 9.2 × 105 RNA copies subjected to the RAT for Omicron-BA.1 compared to 1.3 × 105 to 1.5 × 106 for Omicron-BA.2. Overall, intra-assay differences for the detection of Omicron-BA.1-containing and Omicron-BA.2-containing samples were non-significant, while a marked overall heterogeneity among the five RATs was observed. To score positive in these point-of-care tests, up to 22-fold (LoD50) or 68-fold (LoD95) higher viral loads were required for the worst performing compared to the best performing RAT. The rates of true-positive test results for these Omicron subvariant-containing samples in the highest viral load category (Ct values < 25) ranged between 44.7 and 91.1%, while they dropped to 8.7 to 22.7% for samples with intermediate Ct values (25-30). In light of recent reports on the emergence of two novel Omicron-BA.2 subvariants, Omicron-BA.2.75 and BJ.1, awareness must be increased for the overall reduced detection rate and marked differences in RAT performance for these Omicron subvariants.

Virus variant-specific clinical performance of SARS coronavirus two rapid antigen tests in point-of-care use, from November 2020 to January 2022.

OBJECTIVES: Antigen rapid diagnostic tests (RDTs) for SARS coronavirus 2 (SARS-CoV-2) are quick, widely available, and inexpensive. Consequently, RDTs have been established as an alternative and additional diagnostic strategy to quantitative reverse transcription polymerase chain reaction (RT-qPCR). However, reliable clinical and large-scale performance data specific to a SARS-CoV-2 virus variant of concern (VOC) are limited, especially for the Omicron VOC. The aim of this study was to compare RDT performance among different VOCs. METHODS: This single-centre prospective performance assessment compared RDTs from three manufacturers (NADAL, Panbio, MEDsan) with RT-qPCR including deduced standardized viral load from oropharyngeal swabs for detection of SARS-CoV-2 in a clinical point-of-care setting from November 2020 to January 2022. RESULTS: Among 35 479 RDT/RT-qPCR tandems taken from 26 940 individuals, 164 of the 426 SARS-CoV-2 positive samples tested true positive with an RDT corresponding to an RDT sensitivity of 38.50% (95% CI, 34.00-43.20%), with an overall specificity of 99.67% (95% CI, 99.60-99.72%). RDT sensitivity depended on viral load, with decreasing sensitivity accompanied by descending viral load. VOC-dependent sensitivity assessment showed a sensitivity of 42.86% (95% CI, 32.82-53.52%) for the wild-type SARS-CoV-2, 43.42% (95% CI, 32.86-54.61%) for the Alpha VOC, 37.67% (95% CI, 30.22-45.75%) for the Delta VOC, and 33.67% (95% CI, 25.09-43.49%) for the Omicron VOC. Sensitivity in samples with high viral loads of ≥106 SARS-CoV-2 RNA copies per mL was significantly lower in the Omicron VOC (50.00%; 95% CI, 36.12-63.88%) than in the wild-type SARS-CoV-2 (79.31%; 95% CI, 61.61-90.15%; p 0.015). DISCUSSION: RDT sensitivity for detection of the Omicron VOC is reduced in individuals infected with a high viral load, which curtails the effectiveness of RDTs. This aspect furthert: limits the use of RDTs, although RDTs are still an irreplaceable diagnostic tool for rapid, economic point-of-care and extensive SARS-CoV-2 screening.

Vaccine-hesitant individuals accumulate additional COVID-19 risk due to divergent perception and behaviors related to SARS-CoV-2 testing: a population-based, cross-sectional study.

PURPOSE: To investigate the perception of SARS-CoV-2 detection methods, information sources, and opinions on appropriate behavior after receiving negative or positive test results. METHODS: In a questionnaire-based, cross-sectional study conducted between September 1 and November 17, 2021, epidemiological, behavioral, and COVID-19-related data were acquired from the public in Munich, Germany. RESULTS: Most of the 1388 participants obtained information from online media (82.8%) as well as state and federal authorities (80.3%). 93.4% believed in the accuracy of SARS-CoV-2 PCR testing and 41.2% in the accuracy of rapid antigen tests (RATs). However, RATs were preferred for testing (59.1%) over PCR (51.1%). 24.0% of all individuals were willing to ignore hygiene measures and 76.9% were less afraid of SARS-CoV-2 transmission after receiving a negative PCR test (5.9% and 48.8% in case of a negative RAT). 28.8% reported not to self-isolate after receiving a positive RAT. Multivariate analyses revealed that non-vaccinated individuals relied less on information from governmental authorities (p = 0.0004) and more on social media (p = 0.0216), disbelieved in the accuracy of the PCR test (p ≤ 0.0001) while displaying strong preference towards using RATs (p ≤ 0.0001), were more willing to abandon pandemic-related hygiene measures (p ≤ 0.0001), less afraid of transmitting SARS-CoV-2 after a negative RAT (p ≤ 0.0001), and less likely to isolate after a positive RAT (p ≤ 0.0001). CONCLUSION: Insights into preferred information sources as well as perception, preferences, and behavior related to SARS-CoV-2 testing and hygiene measures are key to refining public health information and surveillance campaigns. Non-vaccinated individuals' divergent believes and behaviors possibly increase their COVID-19 risk.

Quantitative interpretation of PF4/heparin-EIA optical densities in predicting platelet-activating VITT antibodies.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a prothrombotic, heparin-induced thrombocytopenia (HIT)-mimicking, adverse reaction caused by platelet-activating anti-platelet factor 4 (PF4) antibodies that occurs rarely after adenovirus vector-based COVID-19 vaccination. Strength of PF4-dependent enzyme immunoassay (EIA) reactivity-judged by optical density (OD) measurements-strongly predicts platelet-activating properties of HIT antibodies in a functional test. Whether a similar relationship holds for VITT antibodies is unknown. OBJECTIVES: To evaluate probability for positive platelet activation testing for VITT antibodies based upon EIA OD reactivity; and to investigate simple approaches to minimize false-negative platelet activation testing for VITT. METHODS: All samples referred for VITT testing were systematically evaluated by semiquantitative in-house PF4/heparin-EIA (OD readings) and PF4-induced platelet activation (PIPA) testing within a cohort study. EIA-positive sera testing PIPA-negative were retested following 1/4 to 1/10 dilution. Logistic regression was performed to predict the probability of a positive PIPA per magnitude of EIA reactivity. RESULTS: Greater EIA ODs in sera from patients with suspected VITT correlated strongly with greater likelihood of PIPA reactivity. Of 61 sera (with OD values >1.0) testing negative in the PIPA, a high proportion (27/61, 44.3%) became PIPA positive when tested at 1/4 to 1/10 dilution. CONCLUSIONS: VITT serology resembles HIT in that greater EIA OD reactivity predicts higher probability of positive testing for platelet-activating antibodies. Unlike the situation with HIT antibodies, however, diluting putative VITT serum increases probability of a positive platelet activation assay, suggesting that optimal complex formation depends on the stoichiometric ratio of PF4 and anti-PF4 VITT antibodies.

Evidence for increased SARS-CoV-2 susceptibility and COVID-19 severity related to pre-existing immunity to seasonal coronaviruses.

The importance of pre-existing immune responses to seasonal endemic coronaviruses (HCoVs) for the susceptibility to SARS-CoV-2 infection and the course of COVID-19 is the subject of an ongoing scientific debate. Recent studies postulate that immune responses to previous HCoV infections can either have a slightly protective or no effect on SARS-CoV-2 pathogenesis and, consequently, be neglected for COVID-19 risk stratification. Challenging this notion, we provide evidence that pre-existing, anti-nucleocapsid antibodies against endemic α-coronaviruses and S2 domain-specific anti-spike antibodies against ß-coronavirus HCoV-OC43 are elevated in patients with COVID-19 compared to pre-pandemic donors. This finding is particularly pronounced in males and in critically ill patients. Longitudinal evaluation reveals that antibody cross-reactivity or polyclonal stimulation by SARS-CoV-2 infection are unlikely to be confounders. Thus, specific pre-existing immunity to seasonal coronaviruses may increase susceptibility to SARS-CoV-2 and predispose individuals to an adverse COVID-19 outcome, guiding risk management and supporting the development of universal coronavirus vaccines.

Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation.

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

Evidence for increased SARS-CoV-2 susceptibility and COVID-19 severity related to pre-existing immunity to seasonal coronaviruses

Wratil et al. find specific antibody responses against seasonal human coronaviruses, which cause the common cold, to be elevated in patients with COVID-19 compared to pre-pandemic blood donors. This specific immunity is likely pre-existing in patients and increases their susceptibility to SARS-CoV-2 and severity of COVID-19.

Comparison of four commercial, automated antigen tests to detect SARS-CoV-2 variants of concern.

A versatile portfolio of diagnostic tests is essential for the containment of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Besides nucleic acid-based test systems and point-of-care (POCT) antigen (Ag) tests, quantitative, laboratory-based nucleocapsid Ag tests for SARS-CoV-2 have recently been launched. Here, we evaluated four commercial Ag tests on automated platforms and one POCT to detect SARS-CoV-2. We evaluated PCR-positive (n = 107) and PCR-negative (n = 303) respiratory swabs from asymptomatic and symptomatic patients at the end of the second pandemic wave in Germany (February-March 2021) as well as clinical isolates EU1 (B.1.117), variant of concern (VOC) Alpha (B.1.1.7) or Beta (B.1.351), which had been expanded in a biosafety level 3 laboratory. The specificities of automated SARS-CoV-2 Ag tests ranged between 97.0 and 99.7% (Lumipulse G SARS-CoV-2 Ag (Fujirebio): 97.03%, Elecsys SARS-CoV-2 Ag (Roche Diagnostics): 97.69%; LIAISON® SARS-CoV-2 Ag (Diasorin) and SARS-CoV-2 Ag ELISA (Euroimmun): 99.67%). In this study cohort of hospitalized patients, the clinical sensitivities of tests were low, ranging from 17.76 to 52.34%, and analytical sensitivities ranged from 420,000 to 25,000,000 Geq/ml. In comparison, the detection limit of the Roche Rapid Ag Test (RAT) was 9,300,000 Geq/ml, detecting 23.58% of respiratory samples. Receiver-operating-characteristics (ROCs) and Youden's index analyses were performed to further characterize the assays' overall performance and determine optimal assay cutoffs for sensitivity and specificity. VOCs carrying up to four amino acid mutations in nucleocapsid were detected by all five assays with characteristics comparable to non-VOCs. In summary, automated, quantitative SARS-CoV-2 Ag tests show variable performance and are not necessarily superior to a standard POCT. The efficacy of any alternative testing strategies to complement nucleic acid-based assays must be carefully evaluated by independent laboratories prior to widespread implementation.

In-depth profiling of COVID-19 risk factors and preventive measures in healthcare workers.

PURPOSE: To determine risk factors for coronavirus disease 2019 (COVID-19) in healthcare workers (HCWs), characterize symptoms, and evaluate preventive measures against SARS-CoV-2 spread in hospitals. METHODS: In a cross-sectional study conducted between May 27 and August 12, 2020, after the first wave of the COVID-19 pandemic, we obtained serological, epidemiological, occupational as well as COVID-19-related data at a quaternary care, multicenter hospital in Munich, Germany. RESULTS: 7554 HCWs participated, 2.2% of whom tested positive for anti-SARS-CoV-2 antibodies. Multivariate analysis revealed increased COVID-19 risk for nurses (3.1% seropositivity, 95% CI 2.5-3.9%, p = 0.012), staff working on COVID-19 units (4.6% seropositivity, 95% CI 3.2-6.5%, p = 0.032), males (2.4% seropositivity, 95% CI 1.8-3.2%, p = 0.019), and HCWs reporting high-risk exposures to infected patients (5.5% seropositivity, 95% CI 4.0-7.5%, p = 0.0022) or outside of work (12.0% seropositivity, 95% CI 8.0-17.4%, p < 0.0001). Smoking was a protective factor (1.1% seropositivity, 95% CI 0.7-1.8% p = 0.00018) and the symptom taste disorder was strongly associated with COVID-19 (29.8% seropositivity, 95% CI 24.3-35.8%, p < 0.0001). An unbiased decision tree identified subgroups with different risk profiles. Working from home as a preventive measure did not protect against SARS-CoV-2 infection. A PCR-testing strategy focused on symptoms and high-risk exposures detected all larger COVID-19 outbreaks. CONCLUSION: Awareness of the identified COVID-19 risk factors and successful surveillance strategies are key to protecting HCWs against SARS-CoV-2, especially in settings with limited vaccination capacities or reduced vaccine efficacy.

Computational strategies to combat COVID-19: useful tools to accelerate SARS-CoV-2 and coronavirus research.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are free to use and available online, either through web applications or public code repositories. Contact:evbc@unj-jena.de.

Evaluation of two rapid antigen tests to detect SARS-CoV-2 in a hospital setting.

Successful containment strategies for the SARS-CoV-2 pandemic will depend on reliable diagnostic assays. Point-of-care antigen tests (POCT) may provide an alternative to time-consuming PCR tests to rapidly screen for acute infections on site. Here, we evaluated two SARS-CoV-2 antigen tests: the STANDARD™ F COVID-19 Ag FIA (FIA) and the SARS-CoV-2 Rapid Antigen Test (RAT). For diagnostic assessment, we used a large set of PCR-positive and PCR-negative respiratory swabs from asymptomatic and symptomatic patients and health care workers in the setting of two University Hospitals in Munich, Germany, i.e. emergency rooms, patient care units or employee test centers. For FIA, overall clinical sensitivity and specificity were 45.4% (n = 381) and 97.8% (n = 360), respectively, and for RAT, 50.3% (n = 445) and 97.7% (n = 386), respectively. For primary diagnosis of asymptomatic and symptomatic individuals, diagnostic sensitivities were 60.9% (FIA) (n = 189) and 64.5% (RAT) (n = 256). This questions these tests' utility for the reliable detection of acute SARS-CoV-2-infected individuals, in particular in high-risk settings. We support the proposal that convincing high-quality outcome data on the impact of false-negative and false-positive antigen test results need to be obtained in a POCT setting. Moreover, the efficacy of alternative testing strategies to complement PCR assays must be evaluated by independent laboratories, prior to widespread implementation in national and international test strategies.

Hypertension delays viral clearance and exacerbates airway hyperinflammation in patients with COVID-19.

In coronavirus disease 2019 (COVID-19), hypertension and cardiovascular diseases are major risk factors for critical disease progression. However, the underlying causes and the effects of the main anti-hypertensive therapies-angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)-remain unclear. Combining clinical data (n = 144) and single-cell sequencing data of airway samples (n = 48) with in vitro experiments, we observed a distinct inflammatory predisposition of immune cells in patients with hypertension that correlated with critical COVID-19 progression. ACEI treatment was associated with dampened COVID-19-related hyperinflammation and with increased cell intrinsic antiviral responses, whereas ARB treatment related to enhanced epithelial-immune cell interactions. Macrophages and neutrophils of patients with hypertension, in particular under ARB treatment, exhibited higher expression of the pro-inflammatory cytokines CCL3 and CCL4 and the chemokine receptor CCR1. Although the limited size of our cohort does not allow us to establish clinical efficacy, our data suggest that the clinical benefits of ACEI treatment in patients with COVID-19 who have hypertension warrant further investigation.

Host factor prioritization for pan-viral genetic perturbation screens using random intercept models and network propagation.

Genetic perturbation screens using RNA interference (RNAi) have been conducted successfully to identify host factors that are essential for the life cycle of bacteria or viruses. So far, most published studies identified host factors primarily for single pathogens. Furthermore, often only a small subset of genes, e.g., genes encoding kinases, have been targeted. Identification of host factors on a pan-pathogen level, i.e., genes that are crucial for the replication of a diverse group of pathogens has received relatively little attention, despite the fact that such common host factors would be highly relevant, for instance, for devising broad-spectrum anti-pathogenic drugs. Here, we present a novel two-stage procedure for the identification of host factors involved in the replication of different viruses using a combination of random effects models and Markov random walks on a functional interaction network. We first infer candidate genes by jointly analyzing multiple perturbations screens while at the same time adjusting for high variance inherent in these screens. Subsequently the inferred estimates are spread across a network of functional interactions thereby allowing for the analysis of missing genes in the biological studies, smoothing the effect sizes of previously found host factors, and considering a priori pathway information defined over edges of the network. We applied the procedure to RNAi screening data of four different positive-sense single-stranded RNA viruses, Hepatitis C virus, Chikungunya virus, Dengue virus and Severe acute respiratory syndrome coronavirus, and detected novel host factors, including UBC, PLCG1, and DYRK1B, which are predicted to significantly impact the replication cycles of these viruses. We validated the detected host factors experimentally using pharmacological inhibition and an additional siRNA screen and found that some of the predicted host factors indeed influence the replication of these pathogens.