Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic.

BACKGROUND: Over the course of the COVID-19 pandemic, laboratories worldwide have been facing an unprecedented increase in demand for PCR testing because of the high importance of diagnostics for prevention and control of virus spread. Moreover, testing demand has been varying considerably over time, depending on the epidemiological situation, rendering efficient resource allocation difficult. Here, we present a scalable workflow which we implemented in our laboratory to increase PCR testing capacity while maintaining high flexibility regarding the number of samples to be processed. METHODS: We compared the performance of five automated extraction instruments, using dilutions of SARS-CoV-2 cell culture supernatant as well as clinical samples. To increase PCR throughput, we combined the two duplex PCR reactions of our previously published SARS-CoV-2 PCR assay into one quadruplex reaction and compared their limit of detection as well as their performance on the detection of low viral loads in clinical samples. Furthermore, we developed a sample pooling protocol with either two or four samples per pool, combined with a specifically adapted SARS-CoV-2 quadruplex PCR assay, and compared the diagnostic sensitivity of pooled testing and individual testing. RESULTS: All tested automated extraction instruments yielded comparable results regarding the subsequent sensitivity of SARS-CoV-2 detection by PCR. While the limit of detection of the quadruplex SARS-CoV-2 PCR assay (E-Gene assay: 28.7 genome equivalents (ge)/reaction, orf1ab assay: 32.0 ge/reaction) was slightly higher than that of our previously published duplex PCR assays (E-Gene assay: 9.8 ge/reaction, orf1ab assay: 6.6 ge/reaction), the rate of correctly identified positive patient samples was comparable for both assays. Sample pooling with optimized downstream quadruplex PCR showed no loss in diagnostic sensitivity compared to individual testing. CONCLUSION: Specific adaptation of PCR assays can help overcome the potential loss of sensitivity due to higher levels of PCR multiplexing or sample dilution in pooled testing. Combining these adapted PCR assays with different sample processing strategies provides a simple and highly adjustable workflow for resource-efficient SARS-CoV-2 diagnostics. The presented principles can easily be adopted in a variety of laboratory settings as well as be adapted to pathogens other than SARS-CoV-2, making it feasible for any laboratory that conducts PCR diagnostics.

Vaccine effectiveness against severe COVID-19 during the Omicron wave in Germany: results from the COViK study.

PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.

Extensive ITR expansion of the 2022 Mpox virus genome through gene duplication and gene loss.

Poxviruses are known to evolve slower than RNA viruses with only 1-2 mutations/genome/year. Rather than single mutations, rearrangements such as gene gain and loss, which have been discussed as a possible driver for host adaption, were described in poxviruses. In 2022 and 2023 the world is being challenged by the largest global outbreak so far of Mpox virus, and the virus seems to have established itself in the human community for an extended period of time. Here, we report five Mpox virus genomes from Germany with extensive gene duplication and loss, leading to the expansion of the ITR regions from 6400 to up to 24,600 bp. We describe duplications of up to 18,200 bp to the opposed genome end, and deletions at the site of insertion of up to 16,900 bp. Deletions and duplications of genes with functions of supposed immune modulation, virulence and host adaption as B19R, B21R, B22R and D10L are described. In summary, we highlight the need for monitoring rearrangements of the Mpox virus genome rather than for monitoring single mutations only.

Estimating the share of SARS-CoV-2-immunologically naïve individuals in Germany up to June 2022.

After the winter of 2021/2022, the coronavirus disease 2019 (COVID-19) pandemic had reached a phase where a considerable number of people in Germany have been either infected with a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, vaccinated or both, the full extent of which was difficult to estimate, however, because infection counts suffer from under-reporting, and the overlap between the vaccinated and recovered subpopulations is unknown. Yet, reliable estimates regarding population-wide susceptibility were of considerable interest: Since both previous infection and vaccination reduce the risk of severe disease, a low share of immunologically naïve individuals lowers the probability of further severe outbreaks, given that emerging variants do not escape the acquired susceptibility reduction. Here, we estimate the share of immunologically naïve individuals by age group for each of the sixteen German federal states by integrating an infectious-disease model based on weekly incidences of SARS-CoV-2 infections in the national surveillance system and vaccine uptake, as well as assumptions regarding under-ascertainment. We estimate a median share of 5.6% of individuals in the German population have neither been in contact with vaccine nor any variant up to 31 May 2022 (quartile range [2.5%-8.5%]). For the adult population at higher risk of severe disease, this figure is reduced to 3.8% [1.6%-5.9%] for ages 18-59 and 2.1% [1.0%-3.4%] for ages 60 and above. However, estimates vary between German states mostly due to heterogeneous vaccine uptake. Excluding Omicron infections from the analysis, 16.3% [14.1%-17.9%] of the population in Germany, across all ages, are estimated to be immunologically naïve, highlighting the large impact the first two Omicron waves had until the beginning of summer in 2022. The method developed here might be useful for similar estimations in other countries or future outbreaks of other infectious diseases.

Performance of 20 rapid antigen detection tests to detect SARS-CoV-2 B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants using a clinical specimen panel from January 2022, Berlin, Germany.

BackgroundThere are conflicting reports on the performance of rapid antigen detection tests (RDT) in the detection of the SARS-CoV-2 Omicron (B.1.1.529) variant; however, these tests continue to be used frequently to detect potentially contagious individuals with high viral loads.AimThe aim of this study was to investigate comparative detection of the Delta (B.1.617.2) and Omicron variants by using a selection of 20 RDT and a limited panel of pooled combined oro- and nasopharyngeal clinical Delta and Omicron specimens.MethodsWe tested 20 CE-marked RDT for their performance to detect SARS-CoV-2 Delta and Omicron by using a panel of pooled clinical specimens collected in January 2022 in Berlin, Germany.ResultsWe observed equivalent detection performance for Delta and Omicron for most RDT, and sensitivity was widely in line with our previous pre-Delta/Omicron evaluation. Some variation for individual RDT was observed either for Delta vs Omicron detection, or when compared with the previous evaluation, which may be explained both by different panel sizes resulting in different data robustness and potential limitation of batch-to-batch consistency. Additional experiments with three RDT using non-pooled routine clinical samples confirmed comparable performance to detect Delta vs Omicron. Overall, RDT that were previously positively evaluated retained good performance also for Delta and Omicron variants.ConclusionOur findings suggest that currently available RDT are sufficient for the detection of SARS-CoV-2 Delta and Omicron variants.

A large travel-associated outbreak of iatrogenic botulism in four European countries following intragastric botulinum neurotoxin injections for weight reduction, Türkiye, February to March 2023.

In March 2023, 34 associated cases of iatrogenic botulism were detected in Germany (30 cases), Switzerland (two cases), Austria (one case), and France (one case). An alert was rapidly disseminated via European Union networks and communication platforms (Food- and Waterborne Diseases and Zoonoses Network, EpiPulse, Early Warning and Response System) and the International Health Regulation mechanism; the outbreak was investigated in a European collaboration. We traced sources of the botulism outbreak to treatment of weight loss in Türkiye, involving intragastric injections of botulinum neurotoxin. Cases were traced using a list of patients who had received this treatment. Laboratory investigations of the first 12 German cases confirmed nine cases. The application of innovative and highly sensitive endopeptidase assays was necessary to detect minute traces of botulinum neurotoxin in patient sera. The botulism notification requirement for physicians was essential to detect this outbreak in Germany. The surveillance case definition of botulism should be revisited and inclusion of cases of iatrogenic botulism should be considered as these cases might lack standard laboratory confirmation yet warrant public health action. Any potential risks associated with the use of botulinum neurotoxins in medical procedures need to be carefully balanced with the expected benefits of the procedure.

Deep Time Course Proteomics of SARS-CoV- and SARS-CoV-2-Infected Human Lung Epithelial Cells (Calu-3) Reveals Strong Induction of Interferon-Stimulated Gene Expression by SARS-CoV-2 in Contrast to SARS-CoV.

Severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 infections are characterized by remarkable differences, including infectivity and case fatality rate. The underlying mechanisms are not well understood, illustrating major knowledge gaps of coronavirus biology. In this study, protein expression of the SARS-CoV- and SARS-CoV-2-infected human lung epithelial cell line Calu-3 was analyzed using data-independent acquisition-mass spectrometry. This resulted in a comprehensive map of infection-related proteome-wide expression changes in human cells covering the quantification of 7478 proteins across four time points. Most notably, the activation of interferon type-I response was observed, which is surprisingly absent in several proteome studies. The data reveal that SARS-CoV-2 triggers interferon-stimulated gene expression much stronger than SARS-CoV, which reflects the already described differences in interferon sensitivity. Potentially, this may be caused by the enhanced abundance of the viral M protein of SARS-CoV in comparison to SARS-CoV-2, which is a known inhibitor of type I interferon expression. This study expands the knowledge on the host response to SARS-CoV-2 infections on a global scale using an infection model, which seems to be well suited to analyze the innate immunity.

Evaluation of 11 commercially available PCR kits for the detection of monkeypox virus DNA, Berlin, July to September 2022.

Before the international spread of monkeypox in May 2022, PCR kits for the detection of orthopoxviruses, and specifically monkeypox virus, were rarely available. Here we describe the evaluation of 11 recently developed commercially available PCR kits for the detection of monkeypox virus DNA. All tested kits are currently intended for research use only and clinical performance still needs to be assessed in more detail, but all were suitable for diagnostics of monkeypox virus, with variations in specificity rather than sensitivity.

Application of spectral library prediction for parallel reaction monitoring of viral peptides.

A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future.

Inactivation of Coronaviruses during Sample Preparation for Proteomics Experiments.

Mass spectrometry-based proteomics is applied in SARS-CoV-2 research and is, moreover, being discussed as a novel method for SARS-CoV-2 diagnostics. However, the safe inactivation of coronaviruses by proteomics lysis buffers has not been systematically analyzed yet. Hence, for safety reasons a heating step prior to sample preparation is often performed. This step could be omitted once the safe inactivation with the typical buffers is proven. Here we test five different proteomics lysis buffers-4% SDS, 1% SDC, TFA, 6 M GdmCl, and 8 M urea-for their inactivation capacity of coronaviruses. Two representative human coronaviruses, namely HCoV-229E and HCoV-OC43, were used as surrogate for SARS-CoV-2. Lysis was performed at room temperature and at 95 °C for 5 min. Inactivation was confirmed by the absence of a cytopathic effect in MRC-5 cells, and equivocal results were further confirmed by serial passaging and quantitative real-time PCR. While at room temperature SDS, SDC, and TFA inactivated both coronaviruses, and GdmCl and urea resulted in partially incomplete inactivation. This demonstrates that care should be taken when choosing lysis buffers for proteomics analysis of coronaviruses, because some buffers do not ensure inactivation and, hence, biosafety during the further sample preparation.

Replication of cowpox virus in macrophages is dependent on the host range factor p28/N1R.

Zoonotic orthopoxvirus infections continue to represent a threat to human health. The disease caused by distinct orthopoxviruses differs in terms of symptoms and severity, which may be explained by the unique repertoire of virus factors that modulate the host's immune response and cellular machinery. We report here on the construction of recombinant cowpox viruses (CPXV) which either lack the host range factor p28 completely or express truncated variants of p28. We show that p28 is essential for CPXV replication in macrophages of human or mouse origin and that the C-terminal RING finger domain of p28 is necessary to allow CPXV replication in macrophages.

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2.

BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.

Sensitive on-site detection of SARS-CoV-2 by ID NOW COVID-19.

Point of care detection of SARS-CoV-2 is one pillar in a containment strategy and important to break infection chains. Here we report the sensitive, specific and robust detection of SARS-CoV-2 and respective variants of concern by the ID NOW COVID-19 device.

The impact of non-pharmaceutical interventions on COVID-19 epidemic growth in the 37 OECD member states.

We estimated the impact of a comprehensive set of non-pharmeceutical interventions on the COVID-19 epidemic growth rate across the 37 member states of the Organisation for Economic Co-operation and Development during the early phase of the COVID-19 pandemic and between October and December 2020. For this task, we conducted a data-driven, longitudinal analysis using a multilevel modelling approach with both maximum likelihood and Bayesian estimation. We found that during the early phase of the epidemic: implementing restrictions on gatherings of more than 100 people, between 11 and 100 people, and 10 people or less was associated with a respective average reduction of 2.58%, 2.78% and 2.81% in the daily growth rate in weekly confirmed cases; requiring closing for some sectors or for all but essential workplaces with an average reduction of 1.51% and 1.78%; requiring closing of some school levels or all school levels with an average reduction of 1.12% or 1.65%; recommending mask wearing with an average reduction of 0.45%, requiring mask wearing country-wide in specific public spaces or in specific geographical areas within the country with an average reduction of 0.44%, requiring mask-wearing country-wide in all public places or all public places where social distancing is not possible with an average reduction of 0.96%; and number of tests per thousand population with an average reduction of 0.02% per unit increase. Between October and December 2020 work closing requirements and testing policy were significant predictors of the epidemic growth rate. These findings provide evidence to support policy decision-making regarding which NPIs to implement to control the spread of the COVID-19 pandemic.

Establishment of a specimen panel for the decentralised technical evaluation of the sensitivity of 31 rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021.

IntroductionThe detection of SARS-CoV-2 with rapid diagnostic tests (RDT) has become an important tool to identify infected people and break infection chains. These RDT are usually based on antigen detection in a lateral flow approach.AimWe aimed to establish a comprehensive specimen panel for the decentralised technical evaluation of SARS-CoV-2 antigen rapid diagnostic tests.MethodsWhile for PCR diagnostics the validation of a PCR assay is well established, there is no common validation strategy for antigen tests, including RDT. In this proof-of-principle study we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from 1.1 × 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 RDT in up to six laboratories.ResultsOur results show that there is considerable variation in the detection limits and the clinical sensitivity of different RDT. We show that the best RDT can be applied to reliably identify infectious individuals who present with SARS-CoV-2 loads down to 106 genome copies per mL of specimen. For the identification of infected individuals with SARS-CoV-2 loads corresponding to less than 106 genome copies per mL, only three RDT showed a clinical sensitivity of more than 60%.ConclusionsSensitive RDT can be applied to identify infectious individuals with high viral loads but not to identify all infected individuals.

[Crisis management at the Robert Koch Institute during the COVID-19 pandemic and the exchange between federal and state governments]. / Das Lagemanagement des Robert Koch-Instituts während der COVID-19-Pandemie und der Austausch zwischen Bund und Ländern.

The Robert Koch Institute (RKI) plays a central role in Germany in the management of health hazards of biological origin. The RKI's crisis management aims to contribute to protecting the health of the population in Germany in significant epidemic situations and to maintain the RKI's working ability over a long period of time even under high load. This article illustrates the crisis management of the RKI in general as well as during the COVID-19 pandemic. The generic RKI crisis management structures and the setup of the RKI emergency operations centre (EOC), their operationalisation in the context of the COVID-19 pandemic and the resulting challenges as of 31 October 2020 are described in this paper. The exchange between the federal and state governments during the pandemic is also described.The COVID-19 pandemic has led to extraordinary circumstances. During the epidemic situation, good communication and coordination has been essential, both within the RKI and with other federal or state authorities and expert groups. Under great pressure, the RKI produces and regularly updates recommendations, statements and assessments on various topics. To provide operational support for all COVID-19 related activities, an EOC was activated at the RKI. During the COVID-19 pandemic, there are various challenges regarding personnel and structures. It became apparent that good preparation (e.g. existing task descriptions and premises) has an important positive impact on crisis management.

[Measures to cope with the COVID-19 pandemic in Germany: nonpharmaceutical and pharmaceutical interventions]. / Maßnahmen zur Bewältigung der COVID-19-Pandemie in Deutschland: nichtpharmakologische und pharmakologische Ansätze.

When the emerging novel SARS-CoV­2 virus first appeared in December 2019, neither specific therapeutic options nor vaccinations were available. The role of nonpharmaceutical interventions (NPIs) became of central importance. At the Robert Koch Institute, a multilayer strategy consisting of population-based and individual preventive measures to control the pandemic was developed, which built upon existing influenza pandemic plans as well as generic plans. This paper explains the recommended NPIs and illustrates the pharmaceutical approaches developed in parallel.Among others, general contact bans, providing material for infection prevention and control, ban of events, closing educational institutions, and restricting travel are counted among population-based measures. Additional individual preventive measures are necessary, e.g., keeping a minimum distance, reducing contacts, and wearing a mouth-nose covering as well as quarantine and isolation. Measures within the health system are based on recommendations of the Commission on Hospital Hygiene and Infection Protection (Kommission für Krankenhaushygiene und Infektionsprävention (KRINKO)) and specified and implemented by professional societies. Since November 2020, an antiviral therapy with remdesivir and treatment with the glucocorticoid dexamethasone have been available as pharmaceutical interventions. Monoclonal antibodies are at this time not approved. Therapeutic anticoagulation is recommended.Recommendations are constantly adapted to the increasing knowledge on the pathogen and its means of transmission. A challenge is to strengthen the trust of the population. Many measures have to be applied on an individual basis in order to work together.

Perspective on Proteomics for Virus Detection in Clinical Samples.

One of the most widely used methods to detect an acute viral infection in clinical specimens is diagnostic real-time polymerase chain reaction. However, because of the COVID-19 pandemic, mass-spectrometry-based proteomics is currently being discussed as a potential diagnostic method for viral infections. Because proteomics is not yet applied in routine virus diagnostics, here we discuss its potential to detect viral infections. Apart from theoretical considerations, the current status and technical limitations are considered. Finally, the challenges that have to be overcome to establish proteomics in routine virus diagnostics are highlighted.

Serology- and PCR-based cumulative incidence of SARS-CoV-2 infection in adults in a successfully contained early hotspot (CoMoLo study), Germany, May to June 2020.

Three months after a coronavirus disease (COVID-19) outbreak in Kupferzell, Germany, a population-based study (n = 2,203) found no RT-PCR-positives. IgG-ELISA seropositivity with positive virus neutralisation tests was 7.7% (95% confidence interval (CI): 6.5-9.1) and 4.3% with negative neutralisation tests. We estimate 12.0% (95% CI: 10.4-14.0%) infected adults (24.5% asymptomatic), six times more than notified. Full hotspot containment confirms the effectiveness of prompt protection measures. However, 88% naïve adults are still at high COVID-19 risk.