Impact of the COVID-19 pandemic on norovirus circulation in Germany.

Human norovirus is a major cause of viral gastroenteritis in all age groups. The virus is constantly and rapidly changing, allowing mutations and recombination events to create great diversity of circulating viruses. With the start of the COVID-19 pandemic in 2020, a wide range of public health measures were introduced worldwide to control human-to-human transmission of SARS-CoV-2. In Germany, control measures such as distance rules, contact restrictions, personal protection equipment as well as intensive hand hygiene were introduced. To better understand the effect of the measures to control the COVID-19 pandemic on incidence and the molecular epidemiological dynamics of norovirus outbreaks in Germany, we analyzed national notification data between July 2017 and December 2022 and characterized norovirus sequences circulating between January 2018 and December 2022. Compared to a reference period before the pandemic, the incidence of notified norovirus gastroenteritis decreased by 89.7% to 9.6 per 100,000 during the 2020/2021 norovirus season, corresponding to an incidence rate ratio (IRR) of 0.10. Samples from 539 outbreaks were genotyped in two regions of the viral genome from pre-pandemic (January 2018 to February 2020) and samples from 208 outbreaks during pandemic time period (March 2020 to December 2022). As expected, norovirus outbreaks were mainly found in child care facilities and nursing homes. In total, 36 genotypes were detected in the study period. A high proportion of recombinant strains (86%) was found in patients, the proportion of detected recombinant viruses did not vary between the pre-pandemic and pandemic phase. The proportion of the predominant recombinant strain GII.4 Sydney[P16] was unchanged before pandemic and during pandemic at 37.5%. The diversity of most common genotypes in nursing homes and child care facilities showed a different proportion of genotypes causing outbreaks. In nursing homes as well as in child care facilities GII.4 Sydney[P16] was predominant during the whole study period. Compared to the nursing homes, a greater variety of genotypes at the expense of GII.4 Sydney[P16] was detected in child care facilities. Furthermore, the overall proportion of recombinant strain GII.3[P12] increased during the pandemic, due to outbreaks in child care facilities. The COVID-19 pandemic had a high impact on the occurrence of sporadic cases and norovirus outbreaks in Germany, leading to a near suppression of the typical norovirus winter season following the start of the pandemic. The number of norovirus-associated outbreak samples sent to the Consultant Laboratory dropped by 63% during the pandemic. We could not identify a clear influence on circulating norovirus genotypes. The dominance of GII.4 Sydney recombinant strains was independent from the pandemic. Further studies are needed to follow up on the diversity of less predominant genotypes to see if the pandemic could have acted as a bottleneck to the spread of previously minoritized genotypes like GII.3[P12].

Diversity of human astroviruses in Germany 2018 and 2019.

Aim of this study was to investigate the molecular diversity of human astroviruses (HAstV) in Germany. A follow-up study was performed with human stool samples collected in 2018-2019, which were genotyped retrospectively. A total of 2645 stool samples, collected between January 2018 and December 2019 from sporadic cases and outbreaks of acute gastroenteritis were analyzed. An algorithm of PCR systems was used to characterize human astrovirus. Human astroviruses were found in 40 samples (positive rate: 1.6%). During the study period, children aged 1-2 years (48%) were most affected by HAstV. Genotyping revealed a number of nine circulating genotypes representing four human Mamastrovirus species. Strain MLB1 was predominant in the study population with a detection rate of 25% followed by HAstV1 with a positive rate of 20%. The diversity of astrovirus genotypes seems to be rather stable in Germany in the last years. A clustering of regionally and/or temporally linked human astroviruses in Germany was not detectable.

Comprehensive Molecular Approach for Characterization of Hepatitis E Virus Genotype 3 Variants.

Autochthonous hepatitis E virus genotype 3 (HEV-3) infections in industrialized countries are more frequent than previously assumed. HEV-3 is zoonotic and the causal pathogen of chronic hepatitis E. According to the latest classification of the family Hepeviridae, 10 designated HEV-3 subtypes (HEV-3a to HEV-3j) and 7 unassigned HEV-3 subtypes are proposed. In order to identify and characterize the HEV-3 variants in circulation, we developed a molecular approach combining a sensitive HEV-specific real-time reverse transcription-PCR (RT-PCR) targeting the overlapping region of HEV ORF2 and ORF3 (the ORF2/3 region) and two newly designed consensus nested RT-PCRs targeting the HEV ORF1 and ORF2 genes, respectively. Since complete genome sequences are required for new HEV-3 subtype assignment, we implemented a straightforward approach for full-length HEV-3 genome amplification. Twenty-nine human serum samples and six human feces samples from chronic hepatitis E patients were selected for evaluation of the system. Viral loads ranged from 1 × 104 to 1.9 × 1010 copies/ml of serum and from 1.8 × 104 to 1 × 1012 copies/g of feces. Sequence and phylogenetic analyses of partial ORF1 and ORF2 sequences showed that HEV strains had considerable genetic diversity and clustered into the HEV-3c (29/35), HEV-3e (2/35), HEV-3f (2/35), and unassigned HEV-3 (2/35) subtypes. Moreover, from these strains, three full-length HEV-3 genome sequences were generated and characterized. DE/15-0030 represents a typical HEV-3c strain (95.7% nucleotide identity to wbGER27), while DE/15-0031 and SW/16-0282 have <89.2% homology to known HEV-3 strains and are phylogenetically divergent, indicating novel HEV-3 subtypes. In summary, our approach will significantly facilitate the detection, quantification, and determination of HEV-3 strains and will thus help to improve molecular diagnostics and our knowledge of HEV diversity and evolution.

Increased Detection of Emergent Recombinant Norovirus GII.P16-GII.2 Strains in Young Adults, Hong Kong, China, 2016-2017.

A new recombinant norovirus GII.P16-GII.2 outnumbered pandemic GII.4 as the predominant GII genotype in the winter of 2016-2017 in Hong Kong, China. Half of hospitalized case-patients were older children and adults, including 13 young adults. This emergent norovirus targets a wider age population compared with circulating pandemic GII.4 strains.

Differentiation between Wild-Type Group A Rotaviruses and Vaccine Strains in Cases of Suspected Horizontal Transmission and Adverse Events Following Vaccination.

Human group A rotaviruses (RVA) are important enteric pathogens, as they are a leading cause of acute gastroenteritis (AGE) in children worldwide. Since 2013, the German Standing Committee on vaccination recommended the routine rotavirus vaccination for infants in Germany. While vaccination has significantly decreased RVA cases and worldwide mortality, in some cases, infants can develop acute gastroenteritis as an adverse reaction after immunization with an attenuated live vaccine. Pediatricians, as well as clinicians and diagnostic laboratories, contacted the Consultant Laboratory for Rotaviruses and inquired whether cases of RVA-positive AGE after vaccination were associated with vaccine or with wild-type RVA strains. A testing algorithm based on distinguishing PCRs and confirmative sequencing was designed, tested, and applied. Diagnostic samples from 68 vaccinated children and six cases where horizontal transmission was suspected were investigated in this study. Using a combination of real-time PCR, fragment-length analysis of amplicons from multiplex PCRs and confirmative sequencing, vaccine-like virus was detected in 46 samples and wild-type RVA was detected in 6 samples. Three mixed infections of vaccine and wild-type RVA were detectable, no RVA genome was found in 19 samples. High viral loads (>1.0 × 107 copies/g stool) were measured in most RVA-positive samples. Furthermore, information on co-infections with other AGE pathogens in the vaccinated study population was of interest. A commercial multiplex PCR and in-house PCRs revealed three co-infections of vaccinated infants with bacteria (two samples with Clostridioides difficile and one sample with enteropathogenic E. coli) and six co-infections with norovirus in a subset of the samples. Human astrovirus was detected in one sample, with suspected horizontal transmission. The cases of suspected horizontal transmission of vaccine RVA strains could not be confirmed, as they either involved wild-type RVA or were RVA negative. This study shows that RVA-positive AGE after vaccination is not necessarily associated with the vaccine strain and provides a reliable workflow to distinguish RVA vaccine strains from wild-type strains.

Diversity of Noroviruses throughout Outbreaks in Germany 2018.

Human norovirus accounts for the majority of viral gastroenteritis cases worldwide. It is a fast evolving virus generating diversity via mutation and recombination. Therefore, new variants and new recombinant strains emerge in the norovirus population. We characterized norovirus positive stool samples from one intensively studied district Märkisch-Oderland state Brandenburg with the samples from other states of Germany in order to understand the molecular epidemiological dynamics of norovirus outbreaks in Germany 2018. PCR systems, Sanger sequencing, and phylogenetic analyses were used for genotyping. Noroviruses of 250 outbreaks in Germany were genotyped, including 39 outbreaks for the district Märkisch-Oderland. Viral diversity in Märkisch-Oderland as compared to Germany was similar, but not identical. The predominant genogroup in Germany was GII with predominate genotype GII.P16-GII.4 Sydney, whereas GII.P31-GII.4 Sydney was the most frequent in Märkisch-Oderland. Genogroup I viruses were less frequently detected, regional and national. Within the sequences of GII.4 recombinants, two distinct clusters were identified with outbreaks from Märkisch-Oderland. Further analysis of sequence data and detailed epidemiological data are needed in order to understand the link between outbreaks in such clusters. Molecular surveillance should be based on samples collected nationally in order to trace comprehensive virus distribution and recombination events in virus population.

Human astrovirus infection associated with encephalitis in an immunocompetent child: a case report.

BACKGROUND: Until today, classic human astroviruses have not been associated with central nervous system infections in immunocompetent patients. CASE PRESENTATION: A 16-month-old Caucasian girl presented with repetitive generalized seizures with a 4-day history of watery diarrhea, which had already gradually improved. Initially, the prolonged seizures ceased after systemic midazolam treatment and were thought to be fever associated. However, her mental status remained altered, and after seizure recurrence, she was transferred to our pediatric intensive care unit. Seizure control was achieved by a combination of high-dose levetiracetam and phenobarbital, but she remained unconscious. An electroencephalogram at this time revealed generalized high voltage theta activity. All laboratory analyses, including extended blood and cerebrospinal fluid analyses, and a brain magnetic resonance imaging were normal. On day 4, the child gradually became conscious, but was very agitated and not able to walk. Since an electroencephalogram at this time still revealed generalized high voltage theta activity, although she had not received sedative medications for 72 hours, she was diagnosed as having encephalopathy. At that time, results of diagnostic testing of the stool sample were positive for classic astrovirus infection, and we decided to analyze the initially obtained cerebrospinal fluid for astrovirus as well. Cerebrospinal fluid was also found positive for human astrovirus. Sequencing analysis revealed a classic astrovirus genotype 1 with exactly the same nucleotide sequence as in the feces. Clinically, the child gradually improved and was discharged on day 9. CONCLUSIONS: Whereas the new human astrovirus subtypes have been recently associated with central nervous system infection, this is the first case of encephalitis in an immunocompetent child due to classic human astrovirus. Considering that classic human astroviruses are the third most common etiological agents of viral gastroenteritis in children, we believe that human astroviruses as causative agents for central nervous system infections should be considered more often, especially in children and infants with preceding gastroenteritis.

Co-circulation of classic and novel astrovirus strains in patients with acute gastroenteritis in Germany.

OBJECTIVES: In order to analyze the molecular epidemiology of human astroviruses (HAstV) in Germany, a retrospective long-term study was performed to characterize circulating human astrovirus in patients with acute gastroenteritis in Germany. METHODS: A total of 2877 stool samples, collected between January 2010 and December 2015 from sporadic cases and outbreaks of acute gastroenteritis were retrospectively analyzed for astrovirus. A two-step PCR algorithm was developed and used to identify and characterize human astrovirus infections. RESULTS: Overall, 143 samples were astrovirus-positive (5.0%). Astrovirus infection was most frequently detectable in samples from children of 3-4 years (15%) followed by children of 1-2 years (8.6%), detection rates in adults were lower (1%-3.6%). A high number (71.3%) of co-infections, mainly with noro- or rotaviruses, were identified. Genotyping revealed that at least ten genotypes from all four human MAstV species were circulating in the study population. HAstV-1 was predominant in different age groups. Novel HAstV (MLB and VA genotypes) were also circulating in Germany. CONCLUSION: Our findings give new insights into the circulation and genetic diversity of human astroviruses in patients with acute gastroenteritis. The novel HAstV-MLB and -VA genotypes could be characterized firstly in Germany while the analysis showed that these viruses have been dispersed in Germany since 2011 as a causative agent of acute gastroenteritis.

External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014 - 2016 Ebola outbreak in West Africa.

During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied.

External quality assessment studies for laboratory performance of molecular and serological diagnosis of Chikungunya virus infection.

BACKGROUND: Since the re-emergence of Chikungunya virus (CHIKV) in Reunion in 2005 and the recent outbreak in the Caribbean islands with an expansion to the Americas the CHIK diagnostic became very important. OBJECTIVES: We evaluate the performance of laboratories regarding molecular and serological diagnostic of CHIK worldwide. STUDY DESIGN: A panel of 12 samples for molecular and 13 samples for serology were provided to 60 laboratories in 40 countries for evaluating the sensitivity and specificity of molecular and serology testing. RESULTS: The panel for molecular diagnostic testing was analysed by 56 laboratories returning 60 data sets of results whereas the 56 and 60 data sets were returned for IgG and IgM diagnostic from the participating laboratories. Twenty-three from 60 data sets performed optimal, 7 acceptable and 30 sets of results require improvement. From 50 data sets only one laboratory shows an optimal performance for IgM detection, followed by 9 data sets with acceptable and the rest need for improvement. From 46 IgG serology data sets 20 provide an optimal, 2 an acceptable and 24 require improvement performance. The evaluation of some of the diagnostic performances allows linking the quality of results to the in-house methods or commercial assays used. CONCLUSION: The external quality assurance for CHIK diagnostics provides a good overview on the laboratory performance regarding sensitivity and specificity for the molecular and serology diagnostic required for the quick and reliable analysis of suspected CHIK patients. Nearly half of the laboratories have to improve their diagnostic profile to achieve a better performance.

Human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in Germany.

Human metapneumovirus (HMPV) is a cause of respiratory tract illness at all ages. In this study the epidemiological and molecular diversity among patients of different ages was investigated. Between 2000-2001 and 2009-2010, HMPV was detected in 3% (138/4,549) of samples from outpatients with influenza-like illness with a new, sensitive real-time RT-PCR assay. Several hundred (797) clinical specimens from hospitalized children below the age of 4 years with acute respiratory illness were investigated and HMPV was detected in 11.9% of them. Investigation of outpatients revealed that HMPV infections occurred in individuals of all ages but were most prevalent in children (0-4 years) and the elderly (>60 years). The most present clinical features of HMPV infections were cough, bronchitis, fever/shivers and pneumonia. About two thirds of HMPV-positive samples were detected in February and March throughout the study period. Molecular characterization of HMPV revealed a complex cyclic pattern of group dominance where HMPV subgroup A and B viruses predominated in general for three consecutive seasons. German HMPV represented all genetic lineages including A1, A2, B1, B2, sub-clusters A2a and A2b. For Germany, not only time-dependent circulation of lineages and sub-clusters was observed but also co-circulation of two or three predominant lineages. Two newly emerging amino acid substitutions (positions 223 and 280) of lineage B2 were detected in seven German HMPV sequences. Our study gives new insights into the molecular epidemiology of HMPV in in- and outpatients over a time period of 10 years for the first time. It is one of only few long-term surveillance studies in Europe, and allows comparative molecular analyses of HMPV circulating worldwide.

Molecular analysis of human metapneumovirus detected in patients with lower respiratory tract infection in upper egypt.

Introduction. Since 2001, when Human metapneumovirus (HMPV) was isolated in the Netherlands, the virus has been detected in several continents. Although reports have confirmed the prevalence of HMPV worldwide, data from Egypt remain limited. HMPV plays an important role in respiratory tract infections in individuals of all ages particularly in children. This study was aimed at estimating the prevalence of HMPV in patients with community-acquired lower respiratory infection in Upper Egypt and characterizing the circulating Egyptian HMPV strains for the first time. Materials and Methods. From 2005 to 2008, respiratory samples from 520 patients were analyzed for the presence of HMPV by real-time RT-PCR. Molecular and phylogenetic analyses were performed on partial fusion gene sequences of HMPV-positive patients. Results. HMPV-positive patients were detected in 2007-2008. The overall infection rate was 4%, while 57% of the patients were children. Sequence analysis demonstrated circulation of subgroup B viruses with predominance of lineage B2. Nucleotide sequence identity within lineage B1 was 98.8%-99.7% and higher than that in lineage B2 (94.3%-100%). Three new amino acid substitutions (T223N, R229K, and D280N) of lineage B2 were observed. Conclusion. HMPV is a major viral pathogen in the Egyptian population especially in children. During 2007-2008, predominantly HMPV B2 circulated in Upper Egypt.