Atypical age distribution and high disease severity in children with RSV infections during two irregular epidemic seasons throughout the COVID-19 pandemic, Germany, 2021 to 2023.

BackgroundNon-pharmaceutical interventions (NPIs) during the COVID-19 pandemic affected respiratory syncytial virus (RSV) circulation worldwide.AimTo describe, for children aged < 5 years, the 2021 and 2022/23 RSV seasons in Germany.MethodsThrough data and 16,754 specimens from outpatient sentinel surveillance, we investigated RSV seasonality, circulating lineages, and affected children's age distributions in 2021 and 2022/23. Available information about disease severity from hospital surveillance was analysed for patients with RSV-specific diagnosis codes (n = 13,104). Differences between RSV seasons were assessed by chi-squared test and age distributions trends by Mann-Kendall test.ResultsRSV seasonality was irregular in 2021 (weeks 35-50) and 2022/23 (weeks 41-3) compared to pre-COVID-19 2011/12-2019/20 seasons (median weeks 51-12). RSV positivity rates (RSV-PR) were higher in 2021 (40% (522/1,291); p < 0.001) and 2022/23 (30% (299/990); p = 0.005) than in prior seasons (26% (1,430/5,511)). Known globally circulating RSV-A (lineages GA2.3.5 and GA2.3.6b) and RSV-B (lineage GB5.0.5a) strains, respectively, dominated in 2021 and 2022/23. In 2021, RSV-PRs were similar in 1 - < 2, 2 - < 3, 3 - < 4, and 4 - < 5-year-olds. RSV hospitalisation incidence in 2021 (1,114/100,000, p < 0.001) and in 2022/23 (1,034/100,000, p < 0.001) was approximately double that of previous seasons' average (2014/15-2019/20: 584/100,000). In 2022/23, proportions of RSV patients admitted to intensive care units rose (8.5% (206/2,413)) relative to pre-COVID-19 seasons (6.8% (551/8,114); p = 0.004), as did those needing ventilator support (6.1% (146/2,413) vs 3.8% (310/8,114); p < 0.001).ConclusionsHigh RSV-infection risk in 2-4-year-olds in 2021 and increased disease severity in 2022/23 possibly result from lower baseline population immunity, after NPIs diminished exposure to RSV.

A robust, scalable, and cost-efficient approach to whole genome sequencing of RSV directly from clinical samples.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses. IMPORTANCE: Respiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.

Different populations of A(H1N1)pdm09 viruses in a patient with hemolytic-uremic syndrome.

Respiratory viral infections may have different impacts ranging from infection without symptoms to severe disease or even death though the reasons are not well characterized. A patient (age group 5-15 years) displaying symptoms of hemolytic uremic syndrome died one day after hospitalization. qPCR, next generation sequencing, virus isolation, antigenic characterization, resistance analysis was performed and virus replication kinetics in well-differentiated airway cells were determined. Autopsy revealed hemorrhagic pneumonia as major pathological manifestation. Lung samples harbored a large population of A(H1N1)pdm09 viruses with the polymorphism H456H/Y in PB1 polymerase. The H456H/Y viruses replicated much faster to high viral titers than upper respiratory tract viruses in vitro. H456H/Y-infected air-liquid interface cultures of differentiated airway epithelial cells did reflect a more pronounced loss of ciliated cells. A different pattern of virus quasispecies was found in the upper airway samples where substitution S263S/F (HA1) was observed. The data support the notion that viral quasispecies had evolved locally in the lung to support high replicative fitness. This change may have initiated further pathogenic processes leading to rapid dissemination of inflammatory mediators followed by development of hemorrhagic lung lesions and fatal outcome.

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.

Respiratory infections in children and adolescents in Germany during the COVID-19 pandemic.

Background: Before the COVID-19 pandemic, acute respiratory infections (ARIs) in children were mainly characterised by three pathogens: respiratory syncytial viruses (RSV), influenza viruses and rhinoviruses. The impact of the COVID-19 pandemic and the measures taken in Germany (especially until the end of 2021) on the incidence of ARI in children and adolescents aged 0 to 14 years and the pathogens causing them has not yet been comprehensively analysed. Methods: The evaluation is based on data from population-based, virological and hospital-based surveillance instruments up to the end of 2022. Results: After the onset of the COVID-19 pandemic in early 2020, ARI rates remained almost consistently below prepandemic levels until autumn 2021, with only rhinoviruses continuously continuing to cause ARI. Only when the Omicron variant became predominant in 2022, there were measurable COVID-19 rates at population level in children, although COVID-19 hospitalisation rates remained comparatively low. RSV and influenza waves were initially absent and then occurred 'out of season', but were more severe than usual. Conclusions: While the measures taken were effective in inhibiting the number of respiratory infections for almost 1.5 years, moderately frequent but rather mild COVID-19 cases occurred when measures were lifted. When Omicron emerged in 2022 COVID-19 became moderately frequent but led predominantly to mild illnesses. For RSV and influenza, the measures resulted in changes in their annual timing and intensity.

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

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

Human lungs show limited permissiveness for SARS-CoV-2 due to scarce ACE2 levels but virus-induced expansion of inflammatory macrophages.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.

Linking digital surveillance and in-depth virology to study clinical patterns of viral respiratory infections in vulnerable patient populations.

To improve the identification and management of viral respiratory infections, we established a clinical and virologic surveillance program for pediatric patients fulfilling pre-defined case criteria of influenza-like illness and viral respiratory infections. The program resulted in a cohort comprising 6,073 patients (56% male, median age 1.6 years, range 0-18.8 years), where every patient was assessed with a validated disease severity score at the point-of-care using the ViVI ScoreApp. We used machine learning and agnostic feature selection to identify characteristic clinical patterns. We tested all patients for human adenoviruses, 571 (9%) were positive. Adenovirus infections were particularly common and mild in children ≥1 month of age but rare and potentially severe in neonates: with lower airway involvement, disseminated disease, and a 50% mortality rate (n = 2/4). In one fatal case, we discovered a novel virus: HAdV-80. Standardized surveillance leveraging digital technology helps to identify characteristic clinical patterns, risk factors, and emerging pathogens.

Determination of respiratory syncytial virus epidemic seasons by using 95% confidence interval of positivity rates, 2011-2021, Germany.

Based on our national outpatient sentinel surveillance, we have developed a novel approach to determine respiratory syncytial virus (RSV) epidemic seasons in Germany by using RSV positivity rate and its lower limit of 95% confidence interval. This method was evaluated retrospectively on nine RSV seasons, and it is also well-suited to describe off-season circulation of RSV in near real time as observed for seasons 2020/21 and 2021/22 during the COVID-19 pandemic. Prospective application is of crucial importance to enable timely actions for health service delivery and prevention.

Virological Surveillance and Molecular Characterization of Human Parainfluenzavirus Infection in Children with Acute Respiratory Illness: Germany, 2015-2019.

Human parainfluenza viruses (HPIVs) are important causes of respiratory illness, especially in young children. However, surveillance for HPIV is rarely performed continuously, and national-level epidemiologic and genetic data are scarce. Within the German sentinel system, to monitor acute respiratory infections (ARI), 4463 respiratory specimens collected from outpatients < 5 years of age between October 2015 and September 2019 were retrospectively screened for HPIV 1-4 using real-time PCR. HPIV was identified in 459 (10%) samples. HPIV-3 was the most common HPIV-type, with 234 detections, followed by HPIV-1 (113), HPIV-4 (61), and HPIV-2 (49). HPIV-3 was more frequently associated with age < 2 years, and HPIV-4 was more frequently associated with pneumonia compared to other HPIV types. HPIV circulation displayed distinct seasonal patterns, which appeared to vary by type. Phylogenetic characterization clustered HPIV-1 in Clades 2 and 3. Reclassification was performed for HPIV-2, provisionally assigning two distinct HPIV-2 groups and six clades, with German HPIV-2s clustering in Clade 2.4. HPIV-3 clustered in C1, C3, C5, and, interestingly, in A. HPIV-4 clustered in Clades 2.1 and 2.2. The results of this study may serve to inform future approaches to diagnose and prevent HPIV infections, which contribute substantially to ARI in young children in Germany.

Trends in respiratory virus circulation following COVID-19-targeted nonpharmaceutical interventions in Germany, January - September 2020: Analysis of national surveillance data.

BACKGROUND: During the initial COVID-19 response, Germany's Federal Government implemented several nonpharmaceutical interventions (NPIs) that were instrumental in suppressing early exponential spread of SARS-CoV-2. NPI effect on the transmission of other respiratory viruses has not been examined at the national level thus far. METHODS: Upper respiratory tract specimens from 3580 patients with acute respiratory infection (ARI), collected within the nationwide German ARI Sentinel, underwent RT-PCR diagnostics for multiple respiratory viruses. The observation period (weeks 1-38 of 2020) included the time before, during and after a far-reaching contact ban. Detection rates for different viruses were compared to 2017-2019 sentinel data (15350 samples; week 1-38, 11823 samples). FINDINGS: The March 2020 contact ban, which was followed by a mask mandate, was associated with an unprecedented and sustained decline of multiple respiratory viruses. Among these, rhinovirus was the single agent that resurged to levels equalling those of previous years. Rhinovirus rebound was first observed in children, after schools and daycares had reopened. By contrast, other nonenveloped viruses (i.e. gastroenteritis viruses reported at the national level) suppressed after the shutdown did not rebound. INTERPRETATION: Contact restrictions with a subsequent mask mandate in spring may substantially reduce respiratory virus circulation. This reduction appears sustained for most viruses, indicating that the activity of influenza and other respiratory viruses during the subsequent winter season might be low,whereas rhinovirus resurgence, potentially driven by transmission in educational institutions in a setting of waning population immunity, might signal predominance of rhinovirus-related ARIs. FUNDING: Robert Koch-Institute and German Ministry of Health.

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.

Reciprocal circulation pattern of SARS-CoV-2 and influenza viruses during the influenza seasons 2019/2020 and 2020/2021 in the Bavarian Influenza Sentinel (Germany).

The corona virus disease-2019 (COVID-19) pandemic began in Wuhan, China, and quickly spread around the world. The pandemic overlapped with two consecutive influenza seasons (2019/2020 and 2020/2021). This provided the opportunity to study community circulation of influenza viruses and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in outpatients with acute respiratory infections during these two seasons within the Bavarian Influenza Sentinel (BIS) in Bavaria, Germany. From September to March, oropharyngeal swabs collected at BIS were analysed for influenza viruses and SARS-CoV-2 by real-time polymerase chain reaction. In BIS 2019/2020, 1376 swabs were tested for influenza viruses. The average positive rate was 37.6%, with a maximum of over 60% (in January). The predominant influenza viruses were Influenza A(H1N1)pdm09 (n = 202), Influenza A(H3N2) (n = 144) and Influenza B Victoria lineage (n = 129). In all, 610 of these BIS swabs contained sufficient material to retrospectively test for SARS-CoV-2. SARS-CoV-2 RNA was not detectable in any of these swabs. In BIS 2020/2021, 470 swabs were tested for influenza viruses and 457 for SARS-CoV-2. Only three swabs (0.6%) were positive for Influenza, while SARS-CoV-2 was found in 30 swabs (6.6%). We showed that no circulation of SARS-CoV-2 was detectable in BIS during the 2019/2020 influenza season, while virtually no influenza viruses were found in BIS 2020/2021 during the COVID-19 pandemic.

Zoonotic infection with swine A/H1avN1 influenza virus in a child, Germany, June 2020.

A zoonotic A/sw/H1avN1 1C.2.2 influenza virus infection was detected in a German child that presented with influenza-like illness, including high fever. There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.

Correction: The significance of human respiratory syncytial virus (HRSV) in children from Ghana with acute lower respiratory tract infection: A molecular epidemiological analysis, 2006 and 2013-2014.

[This corrects the article DOI: 10.1371/journal.pone.0203788.].