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.

Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management.

In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: ∼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.

Cellular and Humoral SARS-CoV-2 Vaccination Responses in 192 Adult Recipients of Allogeneic Hematopoietic Cell Transplantation.

To determine factors influencing the vaccination response against SARS-CoV-2 is of importance in recipients of allogeneic hematopoietic cell transplantation (allo-HCT) as they display an increased mortality after SARS-CoV-2 infection, an increased risk of extended viral persistence and reduced vaccination response. Real-life data on anti-SARS-CoV-2-S1-IgG titers (n = 192) and IFN-γ release (n = 110) of allo-HCT recipients were obtained using commercially available, validated assays after vaccination with either mRNA (Comirnaty™, Pfizer-BioNTech™, NY, US and Mainz, Germany or Spikevax™, Moderna™, Cambridge, Massachusetts, US) or vector-based vaccines (Vaxzevria™,AstraZeneca™, Cambridge, UK or Janssen COVID-19 vaccine™Johnson/Johnson, New Brunswick, New Jersey, US), or after a heterologous protocol (vector/mRNA). Humoral response (78% response rate) was influenced by age, time after transplantation, the usage of antithymocyte globulin (ATG) and ongoing immunosuppression, specifically corticosteroids. High counts of B cells during the vaccination period correlated with a humoral response. Only half (55%) of participants showed a cellular vaccination response. It depended on age, time after transplantation, ongoing immunosuppression with ciclosporin A, chronic graft-versus-host disease (cGvHD) and vaccination type, with vector-based protocols favoring a response. Cellular response failure correlated with a higher CD8+ count and activated/HLA-DR+ T cells one year after transplantation. Our data provide the basis to assess both humoral and cellular responses after SARS-CoV2 vaccination in daily practice, thereby opening up the possibility to identify patients at risk.

Circulating multimeric immune complexes contribute to immunopathology in COVID-19.

A dysregulated immune response with high levels of SARS-CoV-2 specific IgG antibodies characterizes patients with severe or critical COVID-19. Although a robust IgG response is considered to be protective, excessive triggering of activating Fc-gamma-receptors (FcγRs) could be detrimental and cause immunopathology. Here, we document excessive FcγRIIIA/CD16A activation in patients developing severe or critical COVID-19 but not in those with mild disease. We identify two independent ligands mediating extreme FcγRIIIA/CD16A activation. Soluble circulating IgG immune complexes (sICs) are detected in about 80% of patients with severe and critical COVID-19 at levels comparable to active systemic lupus erythematosus (SLE) disease. FcγRIIIA/CD16A activation is further enhanced by afucosylation of SARS-CoV-2 specific IgG. Utilizing cell-based reporter systems we provide evidence that sICs can be formed prior to a specific humoral response against SARS-CoV-2. Our data suggest a cycle of immunopathology driven by an early formation of sICs in predisposed patients. These findings suggest a reason for the seemingly paradoxical findings of high antiviral IgG responses and systemic immune dysregulation in severe COVID-19. The involvement of circulating sICs in the promotion of immunopathology in predisposed patients opens new possibilities for intervention strategies to mitigate critical COVID-19 progression.

Long COVID symptoms in exposed and infected children, adolescents and their parents one year after SARS-CoV-2 infection: A prospective observational cohort study.

BACKGROUND: Long COVID in children and adolescents remains poorly understood due to a lack of well-controlled studies with long-term follow-up. In particular, the impact of the family context on persistent symptoms following SARS-CoV-2 infection remains unknown. We examined long COVID symptoms in a cohort of infected children, adolescents, and adults and their exposed but non-infected household members approximately 1 year after infection and investigated clustering of persistent symptoms within households. METHODS: 1267 members of 341 households (404 children aged <14 years, 140 adolescents aged 14-18 years and 723 adults) were categorized as having had either a SARS-CoV-2 infection or household exposure to SARS-CoV-2 without infection, based on three serological assays and history of laboratory-confirmed infection. Participants completed questionnaires assessing the presence of long COVID symptoms 11-12 months after infection in the household using online questionnaires. FINDINGS: The prevalence of moderate or severe persistent symptoms was statistically significantly higher in infected than in exposed women (36.4% [95% CI: 30.7-42.4%] vs 14.2% [95% CI: 8.7-21.5%]), infected men (22.9% [95% CI: 17.9-28.5%] vs 10.3% [95% CI: 5.8-16.9%]) and infected adolescent girls (32.1% 95% CI: 17.2-50.5%] vs 8.9% [95%CI: 3.1-19.8%]). However, moderate or severe persistent symptoms were not statistically more common in infected adolescent boys aged 14-18 (9.7% [95% CI: 2.8-23.6%] or in infected children <14 years (girls: 4.3% [95% CI: 1.2-11.0%]; boys: 3.7% [95% CI: 1.1-9.6%]) than in their exposed counterparts (adolescent boys: 0.0% [95% CI: 0.0-6.7%]; girls < 14 years: 2.3% [95% CI: 0·7-6·1%]; boys < 14 years: 0.0% [95% CI: 0.0-2.0%]). The number of persistent symptoms reported by individuals was associated with the number of persistent symptoms reported by their household members (IRR=1·11, p=·005, 95% CI [1.03-1.20]). INTERPRETATION: In this controlled, multi-centre study, infected men, women and adolescent girls were at increased risk of negative outcomes 11-12 months after SARS-CoV-2 infection. Amongst non-infected adults, prevalence of negative outcomes was also high. Prolonged symptoms tended to cluster within families, suggesting family-level interventions for long COVID could prove useful. FUNDING: Ministry of Science, Research and the Arts, Baden-Württemberg, Germany.

Response to SARS-CoV-2 vaccines in patients receiving B-cell modulating antibodies for renal autoimmune disease.

BACKGROUND: Effective SARS-CoV-2 vaccination in patients receiving treatment with B-cell depleting agents is challenging. Information on vaccination responses in these patients are a valuable tool to develop efficient vaccination regimens. METHODS: In this single-center retrospective observational study, we report the humoral and cellular response in 34 patients receiving anti-CD20 antibody treatment for renal immune disease. RESULTS: After base immunization with SARS-CoV-2-vaccines, 92.0% developed a cellular, 32.4% a humoral response. Humoral immunity correlated with B-cell counts and the timespan between anti-CD20 antibody treatment and vaccination. All patients with > 21/µl B-cells, or > 197 days after treatment showed humoral response. CONCLUSIONS: Adequate timing of SARS-CoV-2-vaccinations after anti-CD20 antibody treatment and CD19 measurements are crucial to generate immunity. Awaiting partial B-cell recovery by postponing regularly scheduled anti-CD20 treatment should be considered in patients with stable immune disease. TRIAL REGISTRATION: This study has been retrospectively registered in the German Clinical Trials Register (DRKS00027049) on 29/10/2021.

Case Report: Assessing COVID-19 Transmission in Professional Volleyball in Germany, September to December 2020: An Epidemiological Study.

Introduction: The SARS-CoV-2 pandemic poses extraordinary challenges in all fields of daily life. The WHO recommended social distancing guidelines and person-to-person contact was strongly discouraged to contain transmission. Team-based sports were questioned and widely debated. However, there is a lack of available evidence on the risk of in-game SARS-CoV-2 transmission. We aim to derive new insights regarding the risk of SARS-CoV2 infection during team sports and provide current opinion on how to behave during training and competition. Methods: We report on six competitive male volleyball players (national level) of the same team who were infected with COVID-19. The mode of transmission and potential virus spreading within the team was investigated. The entire course of infection was documented by detailed medical history of the players and RT-PCR tests confirmed suspected infections. Serological testing was performed to establish the antibody status of the team. Results: The investigation did not show strong evidence of viral transmission within the team during sporting activities. Only two players with PCR-proven infection hat detectable antibodies in two antibody assays. Conclusion: Private and social gatherings can spread infection into team sports. Clearly defined test strategies and strict adherence to standard COVID-19 hygiene guidelines during sports seasons cannot be overemphasized.

SARS-CoV-2-specific T-cell epitope repertoire in convalescent and mRNA-vaccinated individuals.

Continuously emerging variants of concern (VOCs) sustain the SARS-CoV-2 pandemic. The SARS-CoV-2 Omicron/B.1.1.529 VOC harbours multiple mutations in the spike protein associated with high infectivity and efficient evasion from humoral immunity induced by previous infection or vaccination. By performing in-depth comparisons of the SARS-CoV-2-specific T-cell epitope repertoire after infection and messenger RNA vaccination, we demonstrate that spike-derived epitopes were not dominantly targeted in convalescent individuals compared to non-spike epitopes. In vaccinees, however, we detected a broader spike-specific T-cell response compared to convalescent individuals. Booster vaccination increased the breadth of the spike-specific T-cell response in convalescent individuals but not in vaccinees with complete initial vaccination. In convalescent individuals and vaccinees, the targeted T-cell epitopes were broadly conserved between wild-type SARS-CoV-2 variant B and Omicron/B.1.1.529. Hence, our data emphasize the relevance of vaccine-induced spike-specific CD8+ T-cell responses in combating VOCs including Omicron/B.1.1.529 and support the benefit of boosting convalescent individuals with mRNA vaccines.

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.

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.

Robust and durable serological response following pediatric SARS-CoV-2 infection.

The quality and persistence of children's humoral immune response following SARS-CoV-2 infection remains largely unknown but will be crucial to guide pediatric SARS-CoV-2 vaccination programs. Here, we examine 548 children and 717 adults within 328 households with at least one member with a previous laboratory-confirmed SARS-CoV-2 infection. We assess serological response at 3-4 months and 11-12 months after infection using a bead-based multiplex immunoassay for 23 human coronavirus antigens including SARS-CoV-2 and its Variants of Concern (VOC) and endemic human coronaviruses (HCoVs), and additionally by three commercial SARS-CoV-2 antibody assays. Neutralization against wild type SARS-CoV-2 and the Delta VOC are analysed in a pseudotyped virus assay. Children, compared to adults, are five times more likely to be asymptomatic, and have higher specific antibody levels which persist longer (96.2% versus 82.9% still seropositive 11-12 months post infection). Of note, symptomatic and asymptomatic infections induce similar humoral responses in all age groups. SARS-CoV-2 infection occurs independent of HCoV serostatus. Neutralization responses of children and adults are similar, although neutralization is reduced for both against the Delta VOC. Overall, the long-term humoral immune response to SARS-CoV-2 infection in children is of longer duration than in adults even after asymptomatic infection.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in Households with Children, Southwest Germany, May-August 2020.

Resolving the role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in households with members from different generations is crucial for containing the current pandemic. We conducted a large-scale, multicenter, cross-sectional seroepidemiologic household transmission study in southwest Germany during May 11-August 1, 2020. We included 1,625 study participants from 405 households that each had ≥1 child and 1 reverse transcription PCR-confirmed SARS-CoV-2-infected index case-patient. The overall secondary attack rate was 31.6% and was significantly higher in exposed adults (37.5%) than in children (24.6%-29.2%; p = <0.015); the rate was also significantly higher when the index case-patient was >60 years of age (72.9%; p = 0.039). Other risk factors for infectiousness of the index case-patient were SARS-CoV-2-seropositivity (odds ratio [OR] 27.8, 95% CI 8.26-93.5), fever (OR 1.93, 95% CI 1.14-3.31), and cough (OR 2.07, 95% CI 1.21-3.53). Secondary infections in household contacts generate a substantial disease burden.

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.

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).

Ad hoc laboratory-based surveillance of SARS-CoV-2 by real-time RT-PCR using minipools of RNA prepared from routine respiratory samples.

BACKGROUND: A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China in late 2019 and subsequently caused a pandemic. Surveillance is important to better appreciate this evolving pandemic and to longitudinally monitor the effectiveness of public health measures. OBJECTIVES: We aimed to provide a rapid, easy to establish and costeffective laboratory-based surveillance tool for SARS-CoV-2. STUDY DESIGN: We used minipools of RNA prepared from nucleic acid extractions of routine respiratory samples. We technically validated the assay and distributed the protocol within an informal network of five German university laboratories. RESULTS: We tested a total of 70 minipools resembling 700 samples shortly before the upsurge of cases in Germany from 17.02.2020 to 10.03.2020. One minipool reacted positive and after resolution one individual sample tested SARS-CoV-2 positive. This sample was from a hospitalized patient not suspected of having contracted SARS-CoV-2. CONCLUSIONS: Our approach of a laboratory-based surveillance for SARSCoV-2 using minipools proved its concept is easily adaptable and resource-saving. It might assist not only public health laboratories in SARS-CoV-2 surveillance.