Virus variant-specific clinical performance of a SARS-CoV-2 rapid antigen test with focus on Omicron variants of concern.

OBJECTIVES: Antigen rapid diagnostic tests (Ag-RDTs) play an important role in the diagnosis of SARS-CoV-2. They are easier, quicker, and less expensive than the 'reference standard' RT-PCR and therefore widely in use. Reliable clinical data with respect to Ag-RDT performance in SARS-CoV-2 Omicron variants of concern (VOCs) are limited. Consequently, the objective of this study was to determine the impact different VOCs-especially Omicron-have on the clinical performance of an Ag-RDT. METHODS: We compared the clinical performance of the Sofia SARS-CoV-2 Ag-RDT to RT-PCR in a real-world, single-centre study in a clinical point-of-care setting in patients admitted to a large hospital via the emergency department from 2 November 2020 to 4 September 2022. RESULTS: Among 38 434 Ag-RDT/RT-PCR tandems taken, 1528 yielded a SARS-CoV-2 positive RT-PCR test result, with a prevalence of 4.0% (95% CI, 3.8-4.2). Overall sensitivity of the Ag-RDT was 63.7% (95% CI, 61.3-66.1) and overall specificity was 99.6% (95% CI, 99.5-99.6). Ag-RDT sensitivity was dependent on viral load (VL), because the sensitivity increased to 93.2% (95% CI, 91.5-94.6) in samples with a VL > 106 SARS-CoV-2 copies/mL. Furthermore, the Ag-RDT was more sensitive in men, and older patients. Variant-dependent sensitivity assessment showed that the sensitivity was significantly lower in Omicron-VOC (64.1%; 95% CI, 60.5-67.6) compared with SARS-CoV-2 wild-type samples (70.0%; 95% CI, 59,8-78,6) (binomial test; p value < 0.001). Analysing the limits of detection showed a 27 times higher 95% limit of detection for the Omicron-VOC BA.5 compared with the SARS-CoV-2 wild-type. DISCUSSION: Ag-RDT sensitivity for detection of patients with lower VLs and with Omicron-VOC is reduced, limiting the effectiveness of Ag-RDTs. However, Ag-RDTs are still an unreplaceable tool for widely available, quick, and inexpensive point-of-care SARS-CoV-2 diagnostics.

Pathogen spectra in hospitalised and nonhospitalised children with community-acquired pneumonia.

Background: Paediatric community-acquired pneumonia (CAP) is a leading cause of paediatric morbidity. However, particularly for outpatients with paediatric CAP, data on aetiology and management are scarce. Methods: The prospective pedCAPNETZ study multicentrically enrols children and adolescents with outpatient-treated or hospitalised paediatric CAP in Germany. Blood and respiratory specimens were collected systematically, and comprehensive analyses of pathogen spectra were conducted. Follow-up evaluations were performed until day 90 after enrolment. Results: Between December 2014 and August 2020, we enrolled 486 children with paediatric CAP at eight study sites, 437 (89.9%) of whom had radiographic evidence of paediatric CAP. Median (interquartile range) age was 4.5 (1.6-6.6) years, and 345 (78.9%) children were hospitalised. The most prevalent symptoms at enrolment were cough (91.8%), fever (89.2%) and tachypnoea (62.0%). Outpatients were significantly older, displayed significantly lower C-reactive protein levels and were significantly more likely to be symptom-free at follow-up days 14 and 90. Pathogens were detected in 90.3% of all patients (one or more viral pathogens in 68.1%; one or more bacterial strains in 18.7%; combined bacterial/viral pathogens in 4.1%). Parainfluenza virus and Mycoplasma pneumoniae were significantly more frequent in outpatients. The proportion of patients with antibiotic therapy was comparably high in both groups (92.4% of outpatients versus 86.2% of hospitalised patients). Conclusion: We present first data on paediatric CAP with comprehensive analyses in outpatients and hospitalised cases and demonstrate high detection rates of viral pathogens in both groups. Particularly in young paediatric CAP patients with outpatient care, antibiotic therapy needs to be critically debated.

Total escape of SARS-CoV-2 from dual monoclonal antibody therapy in an immunocompromised patient.

Monoclonal antibodies (mAbs) directed against the spike of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective therapeutic options to combat infections in high-risk patients. Here, we report the adaptation of SARS-CoV-2 to the mAb cocktail REGN-COV in a kidney transplant patient with hypogammaglobulinemia. Following mAb treatment, the patient did not clear the infection. During viral persistence, SARS-CoV-2 acquired three novel spike mutations. Neutralization and mouse protection analyses demonstrate a complete viral escape from REGN-COV at the expense of ACE-2 binding. Final clearance of the virus occurred upon reduction of the immunosuppressive regimen and total IgG substitution. Serology suggests that the development of highly neutralizing IgM rather than IgG substitution aids clearance. Our findings emphasise that selection pressure by mAbs on SARS-CoV-2 can lead to development of escape variants in immunocompromised patients. Thus, modification of immunosuppressive therapy, if possible, might be preferable to control and clearance of the viral infection.

Boosting compromised SARS-CoV-2-specific immunity with mRNA vaccination in liver transplant recipients.

BACKGROUND & AIMS: Liver transplant recipients (LTRs) demonstrate a reduced response to COVID-19 mRNA vaccination; however, a detailed understanding of the interplay between humoral and cellular immunity, especially after a third (and fourth) vaccine dose, is lacking. METHODS: We longitudinally compared the humoral, as well as CD4+ and CD8+ T-cell, responses between LTRs (n = 24) and healthy controls (n = 19) after three (LTRs: n = 9 to 16; healthy controls: n = 9 to 14 per experiment) to four (LTRs: n = 4; healthy controls: n = 4) vaccine doses, including in-depth phenotypical and functional characterization. RESULTS: Compared to healthy controls, development of high antibody titers required a third vaccine dose in most LTRs, while spike-specific CD8+ T cells with robust recall capacity plateaued after the second vaccine dose, albeit with a reduced frequency and epitope repertoire compared to healthy controls. This overall attenuated vaccine response was linked to a reduced frequency of spike-reactive follicular T helper cells in LTRs. CONCLUSION: Three doses of a COVID-19 mRNA vaccine induce an overall robust humoral and cellular memory response in most LTRs. Decisions regarding additional booster doses may thus be based on individual vaccine responses as well as evolution of novel variants of concern. IMPACT AND IMPLICATIONS: Due to immunosuppressive medication, liver transplant recipients (LTR) display reduced antibody titers upon COVID-19 mRNA vaccination, but the impact on long-term immune memory is not clear. Herein, we demonstrate that after three vaccine doses, the majority of LTRs not only exhibit substantial antibody titers, but also a robust memory T-cell response. Additional booster vaccine doses may be of special benefit for a small subset of LTRs with inferior vaccine response and may provide superior protection against evolving novel viral variants. These findings will help physicians to guide LTRs regarding the benefit of booster vaccinations.

Assessing severe acute respiratory syndrome coronavirus 2 infectivity by reverse-transcription polymerase chain reaction: A systematic review and meta-analysis.

The cornerstone of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is reverse-transcription polymerase chain reaction (RT-PCR) of viral RNA. As a surrogate assay SARS-CoV-2 RNA detection does not necessarily imply infectivity. Only virus isolation in permissive cell culture systems can indicate infectivity. Here, we review the evidence on RT-PCR performance in detecting infectious SARS-CoV-2. We searched for any studies that used RT-PCR and cell culture to determine infectious SARS-CoV-2 in respiratory samples. We assessed (i) diagnostic accuracy of RT-PCR compared to cell culture as reference test, (ii) performed meta-analysis of positive predictive values (PPV) and (iii) determined the virus isolation probabilities depending on cycle threshold (Ct) or log10 genome copies/ml using logistic regression. We included 55 studies. There is substantial statistical and clinical heterogeneity. Seven studies were included for diagnostic accuracy. Sensitivity ranged from 90% to 99% and specificity from 29% to 92%. In meta-analysis, the PPVs varied across subgroups with different sampling times after symptom onset, with 1% (95% confidence interval [CI], 0%-7%) in sampling beyond 10 days and 27% (CI, 19%-36%) to 46% (CI, 33%-60%) in subgroups that also included earlier samples. Estimates of virus isolation probability varied between 6% (CI, 0%-100%) and 50% (CI, 0%-100%) at a Ct value of 30 and between 0% (CI, 0%-22%) and 63% (CI, 0%-100%) at 5 log10 genome copies/ml. Evidence on RT-PCR performance in detecting infectious SARS-CoV-2 in respiratory samples was limited. Major limitations were heterogeneity and poor reporting. RT-PCR and cell culture protocols need further standardisation.

Antibody escape and global spread of SARS-CoV-2 lineage A.27.

In spring 2021, an increasing number of infections was observed caused by the hitherto rarely described SARS-CoV-2 variant A.27 in south-west Germany. From December 2020 to June 2021 this lineage has been detected in 31 countries. Phylogeographic analyses of A.27 sequences obtained from national and international databases reveal a global spread of this lineage through multiple introductions from its inferred origin in Western Africa. Variant A.27 is characterized by a mutational pattern in the spike gene that includes the L18F, L452R and N501Y spike amino acid substitutions found in various variants of concern but lacks the globally dominant D614G. Neutralization assays demonstrate an escape of A.27 from convalescent and vaccine-elicited antibody-mediated immunity. Moreover, the therapeutic monoclonal antibody Bamlanivimab and partially the REGN-COV2 cocktail fail to block infection by A.27. Our data emphasize the need for continued global monitoring of novel lineages because of the independent evolution of new escape mutations.

Performance of the Sofia SARS-CoV-2 rapid antigen test as frontline test in a university hospital, Germany.

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of high importance for individual patient care and hospital infection prevention. We aimed to evaluate the performance of the Sofia SARS-CoV-2 antigen rapid diagnostic test (Ag-RDT) in comparison to real-time reverse-transcription polymerase chain reaction (RT-PCR). We conducted a prospective, monocentric cross-sectional study in an emergency department of a German university hospital from November 2020 to March 2021. We tested all samples using both Sofia SARS-CoV-2 Ag-RDT and real-time RT-PCR. A total of 7877 patients were included. Overall sensitivity of the Ag-RDT was 62.9% and specificity was 99.4%. Sensitivity varied across study months, whereas specificity remained high. Sensitivity increased to 94.2% in samples with a cycle threshold (Ct)-value ≤25. The Sofia Ag-RDT proved to be a rapid tool to detect samples with high viral loads (Ct-value ≤25) and might thus help to identify infectious patients.

Accuracy and real life performance of a novel interferon-γ release assay for the detection of SARS-CoV2 specific T cell response.

BACKGROUND: The reliable detection of T cell response to COVID-19 or COVID-19 vaccination is important for individual patient care and for monitoring the immune response e.g. in COVID-19 vaccine trials in a standardized fashion. OBJECTIVES AND STUDY DESIGN: We used blood samples from health care workers (HCW) with or without history of COVID-19 to define test accuracy of a novel interferon-γ release assay (IGRA). For a real-life performance evaluation, we analysed interferon-γ response to complete COVID-19 vaccination in HCW receiving homologous or heterologous vaccination regimens and in patients receiving immunosuppressive or immune modulating therapies. RESULTS: The assay had a specificity of 100%. Sensitivity of the IGRA to detect past infection was 72.2% after infection more than 5 months ago and 93.8% after COVID-19 up to 5 months ago. Quantitative results showed significant differences between first and second vaccine dose, but no difference between homologous and heterologous vaccination regimen. Immunocompromised patients often had no immune response or isolated T cell or antibody response to complete vaccination. CONCLUSIONS: The novel IGRA proved to be a highly specific tool to detect SARS-CoV-2 specific T cell response to COVID-19 as well as COVID-19 vaccination, with sensitivity getting lower over time. In perspective, it may serve as a standardized tool in COVID-19 vaccine trials and in clinical care of immunosuppressed patients.

Case Series: Convalescent Plasma Therapy for Patients with COVID-19 and Primary Antibody Deficiency.

Patients with primary antibody deficiency are at risk for severe and in many cases for prolonged COVID-19. Convalescent plasma treatment of immunocompromised individuals could be an option especially in countries with limited access to monoclonal antibody therapies. While studies in immunocompetent COVID19 patients have demonstrated only a limited benefit, evidence for the safety, timing, and effectiveness of this treatment in antibody-deficient patients is lacking. Here, we describe 16 cases with primary antibody deficiency treated with convalescent plasma in four medical centers. In our cohort, treatment was associated with a reduction in viral load and improvement of clinical symptoms, even when applied over a week after onset of infection. There were no relevant side effects besides a short-term fever reaction in one patient. Longitudinal full-genome sequencing revealed the emergence of mutations in the viral genome, potentially conferring an antibody escape in one patient with persistent viral RNA shedding upon plasma treatment. However, he resolved the infection after a second course of plasma treatment. Thus, our data suggest a therapeutic benefit of convalescent plasma treatment in patients with primary antibody deficiency even months after infection. While it appears to be safe, PCR follow-up for SARS-CoV-2 is advisable and early re-treatment might be considered in patients with persistent viral shedding.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient as a source of immune escape variants.

The origin of SARS-CoV-2 variants of concern remains unclear. Here, we test whether intra-host virus evolution during persistent infections could be a contributing factor by characterizing the long-term SARS-CoV-2 infection dynamics in an immunosuppressed kidney transplant recipient. Applying RT-qPCR and next-generation sequencing (NGS) of sequential respiratory specimens, we identify several mutations in the viral genome late in infection. We demonstrate that a late viral isolate exhibiting genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil, can escape neutralization by COVID-19 antisera. Moreover, infection of susceptible mice with this patient's escape variant elicits protective immunity against re-infection with either the parental virus and the escape variant, as well as high neutralization titers against the alpha and beta SARS-CoV-2 variants, B.1.1.7 and B.1.351, demonstrating a considerable immune control against such variants of concern. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results suggest that immunocompromised patients could be a source for the emergence of potentially harmful SARS-CoV-2 variants.

Evaluation of a multiplex PCR screening approach to identify community-acquired bacterial co-infections in COVID-19: a multicenter prospective cohort study of the German competence network of community-acquired pneumonia (CAPNETZ).

PURPOSE: Thorough knowledge of the nature and frequency of co-infections is essential to optimize treatment strategies and risk assessment in cases of coronavirus disease 2019 (COVID-19). This study aimed to evaluate the multiplex polymerase chain reaction (PCR) screening approach for community-acquired bacterial pathogens (CABPs) at hospital admission, which could facilitate identification of bacterial co-infections in hospitalized COVID-19 patients. METHODS: Clinical data and biomaterials from 200 hospitalized COVID-19 patients from the observational cohort of the Competence Network for community-acquired pneumonia (CAPNETZ) prospectively recruited between March 17, 2020, and March 12, 2021 in 12 centers in Germany and Switzerland, were included in this study. Nasopharyngeal swab samples were analyzed on hospital admission using multiplex real-time reverse transcription (RT)-PCR for a broad range of CABPs. RESULTS: In total of 200 patients Staphylococcus aureus (27.0%), Haemophilus influenzae (13.5%), Streptococcus pneumoniae (5.5%), Moraxella catarrhalis (2.5%), and Legionella pneumophila (1.5%) were the most frequently detected bacterial pathogens. PCR detection of bacterial pathogens correlated with purulent sputum, and showed no correlation with ICU admission, mortality, and inflammation markers. Although patients who received antimicrobial treatment were more often admitted to the ICU and had a higher mortality rate, PCR pathogen detection was not significantly related to antimicrobial treatment. CONCLUSION: General CABP screening using multiplex PCR with nasopharyngeal swabs may not facilitate prediction or identification of bacterial co-infections in the early phase of COVID-19-related hospitalization. Most patients with positive PCR results appear to be colonized rather than infected at that time, questioning the value of routine antibiotic treatment on admission in COVID-19 patients.

The impact of the SARS-CoV-2 pandemic on the prevalence of respiratory tract pathogens in patients with community-acquired pneumonia in Germany.

We show a shift in the prevalence of respiratory viral pathogens in community-acquired pneumonia patients during the COVID-19 pandemic. Our data support the efficiency of non-pharmaceutical interventions on virus circulation except for rhinoviruses. The consequences of an altered circulation on subsequent winter seasons remain unclear and support the importance of systematic virological surveillance.

Characterization of pre-existing and induced SARS-CoV-2-specific CD8+ T cells.

Emerging data indicate that SARS-CoV-2-specific CD8+ T cells targeting different viral proteins are detectable in up to 70% of convalescent individuals1-5. However, very little information is currently available about the abundance, phenotype, functional capacity and fate of pre-existing and induced SARS-CoV-2-specific CD8+ T cell responses during the natural course of SARS-CoV-2 infection. Here, we define a set of optimal and dominant SARS-CoV-2-specific CD8+ T cell epitopes. We also perform a high-resolution ex vivo analysis of pre-existing and induced SARS-CoV-2-specific CD8+ T cells, applying peptide-loaded major histocompatibility complex class I (pMHCI) tetramer technology. We observe rapid induction, prolonged contraction and emergence of heterogeneous and functionally competent cross-reactive and induced memory CD8+ T cell responses in cross-sectionally analyzed individuals with mild disease following SARS-CoV-2 infection and three individuals longitudinally assessed for their T cells pre- and post-SARS-CoV-2 infection. SARS-CoV-2-specific memory CD8+ T cells exhibited functional characteristics comparable to influenza-specific CD8+ T cells and were detectable in SARS-CoV-2 convalescent individuals who were seronegative for anti-SARS-CoV-2 antibodies targeting spike (S) and nucleoprotein (N). These results define cross-reactive and induced SARS-CoV-2-specific CD8+ T cell responses as potentially important determinants of immune protection in mild SARS-CoV-2 infection.

Prevalence of SARS-CoV-2 Infection in Children and Their Parents in Southwest Germany.

Importance: School and daycare closures were enforced as measures to confine the novel coronavirus disease 2019 (COVID-19) pandemic, based on the assumption that young children may play a key role in severe acute respiratory coronavirus 2 (SARS-CoV-2) spread. Given the grave consequences of contact restrictions for children, a better understanding of their contribution to the COVID-19 pandemic is of great importance. Objective: To describe the rate of SARS-CoV-2 infections and the seroprevalence of SARS-CoV-2 antibodies in children aged 1 to 10 years, compared with a corresponding parent of each child, in a population-based sample. Design, Setting, and Participants: This large-scale, multicenter, cross-sectional investigation (the COVID-19 BaWü study) enrolled children aged 1 to 10 years and a corresponding parent between April 22 and May 15, 2020, in southwest Germany. Exposures: Potential exposure to SARS-CoV-2. Main Outcomes and Measures: The main outcomes were infection and seroprevalence of SARS-CoV-2. Participants were tested for SARS-CoV-2 RNA from nasopharyngeal swabs by reverse transcription-polymerase chain reaction and SARS-CoV-2 specific IgG antibodies in serum by enzyme-linked immunosorbent assays and immunofluorescence tests. Discordant results were clarified by electrochemiluminescence immunoassays, a second enzyme-linked immunosorbent assay, or an in-house Luminex-based assay. Results: This study included 4964 participants: 2482 children (median age, 6 [range, 1-10] years; 1265 boys [51.0%]) and 2482 parents (median age, 40 [range, 23-66] years; 615 men [24.8%]). Two participants (0.04%) tested positive for SARS-CoV-2 RNA. The estimated SARS-CoV-2 seroprevalence was low in parents (1.8% [95% CI, 1.2-2.4%]) and 3-fold lower in children (0.6% [95% CI, 0.3-1.0%]). Among 56 families with at least 1 child or parent with seropositivity, the combination of a parent with seropositivity and a corresponding child with seronegativity was 4.3 (95% CI, 1.19-15.52) times higher than the combination of a parent who was seronegative and a corresponding child with seropositivity. We observed virus-neutralizing activity for 66 of 70 IgG-positive serum samples (94.3%). Conclusions and Relevance: In this cross-sectional study, the spread of SARS-CoV-2 infection during a period of lockdown in southwest Germany was particularly low in children aged 1 to 10 years. Accordingly, it is unlikely that children have boosted the pandemic. This SARS-CoV-2 prevalence study, which appears to be the largest focusing on children, is instructive for how ad hoc mass testing provides the basis for rational political decision-making in a pandemic.

COVID-19 in a Severely Immunosuppressed Patient With Life-Threatening Eosinophilic Granulomatosis With Polyangiitis.

Immunosuppressive therapies increase the susceptibility of patients to infections. The current pandemic with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compels clinicians to develop recommendations for successful clinical management and surveillance of immunocompromised patients at high risk for severe disease progression. With only few case studies published on SARS-CoV-2 infection in patients with rheumatic diseases, we report a 25-year-old male who developed moderate coronavirus disease 2019 (COVID-19) with fever, mild dyspnea, and no major complications despite having received high-dose prednisolone, cyclophosphamide, and rituximab for the treatment of highly active, life-threatening eosinophilic granulomatosis with polyangiitis (EGPA).