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Since the COVID-19 pandemic began, wastewater-based surveillance has been used to track circulation levels of SARS-CoV-2 (1,2). For that purpose, we began collecting samples from a regional wastewater treatment plant [...]

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021–2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0–5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages. [ABSTRACT FROM AUTHOR]

During the Belgian winter and spring season 2022–2023, we investigated the potential of used paper tissue (UPT) as a noninvasive sampling method for the diagnosis of acute respiratory infections. Screening for respiratory pathogens was done using an in‐house developed respiratory panel for simultaneous detection of 22 respiratory viruses and seven nonviral pathogens. The method allowed the identification and typing of respiratory pathogens in symptomatic individuals, as well as in collective samples taken at a community level. Pathogens that were identified in nasal swabs could also be detected in concurrent UPT from the same patient. In all cases that tested positive on an antigen‐detection rapid diagnostic test, the corresponding virus could be detected in UPT. The collection of UPT could be useful in epidemiological surveillance of severe acute respiratory syndrome coronavirus 2 and other coronaviruses, as well as other respiratory pathogens such as influenzavirus, respiratory syncytial virus, entero/rhinoviruses including EV‐D68, parainfluenzaviruses, and Streptococcus pneumoniae. Multiple respiratory pathogens could be detected in UPTs of collectivities, confirming its applicability for community testing. This is especially interesting for screening in nursing homes, centers for the disabled, schools or other settings were taking nasal or nasopharyngeal samples is cumbersome. [ABSTRACT FROM AUTHOR]

Wastewater surveillance plays an important role in the management of the coronavirus disease 2019 (COVID‐19) pandemic all over the world. Using different wastewater collection points in Leuven, we wanted to investigate the use of wastewater surveillance as an early warning system for an uprise of infections and as a tool to follow the circulation of specific variants of concern (VOCs) in particular geographic areas. Wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. After virus concentration, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA was quantified by real‐time quantitative polymerase chain reaction (RT‐qPCR) and normalized with the human fecal indicator pepper mild mottle virus (PMMoV). A combination of multiplex RT‐qPCR assays was used to detect signature mutations of circulating VOCs. Fecal virus shedding of SARS‐CoV‐2 variants was measured in feces samples of hospitalized patients. In two residential sampling sites, a rise in wastewater SARS‐CoV‐2 concentration preceded peaks in positive cases. In the WWTP, viral load peaks were seen concomitant with the consecutive waves of positive cases caused by the original Wuhan SARS‐CoV‐2 strain and subsequent VOCs. During the Omicron BA.1 wave, the wastewater viral load increased to a lesser degree, even after normalization of SARS‐CoV‐2 concentration using PMMoV. This might be attributable to a lower level of fecal excretion of this variant. Circulation of SARS‐CoV‐2 VOCs Alpha, Delta, Omicron BA1/BA.2, and BA.4/BA.5 could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. Wastewater‐based surveillance is a sensitive tool to monitor SARS‐CoV‐2 circulation levels and VOCs in larger regions. In times of reduced test capacity, this can prove to be highly valuable. Differences in excretion levels of various SARS‐CoV‐2 variants should however be taken into account when using wastewater surveillance to monitor SARS‐CoV‐2 circulation levels in the population. [ABSTRACT FROM AUTHOR]

• Rotavirus vaccine-derived AGE cases were detected in Belgium in the post-vaccine era. • There was limited evidence for vaccine strain circulation with scarce point mutations in both shed and circulating strains. • 90% of the mutations were non-synonymous and several amino acid substitutions were shared among patients. • Some substitutions could be revertants towards the unattenuated vaccine precursor strain, albeit with low safety concerns. • In 35% of the cases, the AGE was estimated to be of non-rotavirus etiology. Since the introduction of live-attenuated rotavirus vaccines in Belgium in 2006, surveillance has routinely detected rotavirus vaccine-derived strains. However, their genomic landscape and potential role in gastroenteritis have not been thoroughly investigated. We compared VP7 and VP4 nucleotide sequences obtained from rotavirus surveillance with the Rotarix vaccine sequence. As a result, we identified 80 vaccine-derived strains in 5125 rotavirus-positive infants with gastroenteritis from 2007 to 2018. Using both viral metagenomics and reverse transcription qPCR, we evaluated the vaccine strains and screened for co-infecting enteropathogens. Among the 45 patients with known vaccination status, 39 were vaccinated and 87% received the vaccine less than a month before the gastroenteritis episode. Reconstruction of 30 near complete vaccine-derived genomes revealed 0–11 mutations per genome, with 88% of them being non-synonymous. This, in combination with several shared amino acid changes among strains, pointed at selection of minor variant(s) present in the vaccine. We also found that some of these substitutions were true revertants (e.g., F167L on VP4, and I45T on NSP4). Finally, co-infections with known (e.g., Clostridioides difficile and norovirus) and divergent or emerging (e.g., human parechovirus A1, salivirus A2) pathogens were detected, and we estimated that 35% of the infants likely had gastroenteritis due to a 'non-rotavirus' cause. Conversely, we could not rule out the vaccine-derived gastroenteritis in over half of the cases. Continued studies inspecting reversion to pathogenicity should monitor the long-time safety of live-attenuated rotavirus vaccines. All in all, the complementary approach with NGS and qPCR provided a better understanding of rotavirus vaccine strain evolution in the Belgian population and epidemiology of co-infecting enteropathogens in suspected rotavirus vaccine-derived gastroenteritis cases. [ABSTRACT FROM AUTHOR]

We report two clusters of SARS-CoV-2 B.1.617.2 (Delta variant) infections in a group of 41 Indian nursing students who travelled from New Delhi, India, to Belgium via Paris, France. All students tested negative before departure and had a second negative antigen test upon arrival in Paris. Upon arrival in Belgium, the students were quarantined in eight different houses. Four houses remained COVID-free during the 24 days of follow-up, while all 27 residents of the other four houses developed an infection during quarantine, including the four residents who were fully vaccinated and the two residents who were partially vaccinated. Genome sequencing revealed two distinct clusters affecting one and three houses, respectively. In this group of students, vaccination status did not seem to prevent infection nor decrease the viral load. No severe symptoms were reported. Extensive contact tracing and 3 months of nationwide genomic surveillance confirmed that these outbreaks were successfully contained and did not contribute to secondary community transmission in Belgium. These clusters highlight the importance of repeated testing and quarantine measures among travelers coming from countries experiencing a surge of infections, as all infections were detected 6 days or more after arrival. [ABSTRACT FROM AUTHOR]

Because of the enormous variation in their genome sequence, genotyping enteroviruses by standard methods can prove to be quite challenging. Nanopore sequencing offers the potential to overcome the limitations of older techniques, but thus far, only amplicon-based strategies have been used to sequence complete enterovirus genomes. By combining a sequence-independent, single primer amplification (SISPA) for cDNA generation with next-generation sequencing using the Oxford Nanopore MinION, complete enterovirus genomes can be obtained in an easy-to-use, sequence-independent manner. To demonstrate its usability, we applied this technique to determine the complete genome sequence of an enterovirus C104 strain, representing the first documented occurrence of this uncommon enterovirus strain in Belgium. • Sequence-independent Nanopore sequencing using the Oxford Nanopore MinION. • First detection and complete genome characterization of an enterovirus C104 in Belgium. • Easy-to-use complete genome sequencing with a fast turnaround time. [ABSTRACT FROM AUTHOR]

Coronavirus Disease 2019 (COVID-19) vaccination has resulted in excellent protection against fatal disease, including in older adults. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases among residents) by combining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aerosol monitoring, whole-genome phylogenetic analysis and immunovirological profiling of nasal mucosa by digital nCounter transcriptomics. Phylogenetic investigations indicated that each outbreak stemmed from a single introduction event, although with different variants (Delta, Gamma and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 d after the initial infection. Combining demographic, immune and viral parameters, the best predictive models for mortality comprised IFNB1 or age, viral ORF7a and ACE2 receptor transcripts. Comparison with published pre-vaccine fatal COVID-19 transcriptomic and genomic signatures uncovered a unique IRF3 low/IRF7 high immune signature in post-vaccine fatal COVID-19 outbreaks. A multi-layered strategy, including environmental sampling, immunomonitoring and early antiviral therapy, should be considered to prevent post-vaccination COVID-19 mortality in nursing homes., (© 2023. The Author(s).)

BackgroundLateral flow antigen-detection rapid diagnostic tests (Ag-RDTs) for viral infections constitute a fast, cheap and reliable alternative to nucleic acid amplification tests (NAATs). Whereas leftover material from NAATs can be employed for genomic analysis of positive samples, there is a paucity of information on whether viral genetic characterisation can be achieved from archived Ag-RDTs.AimTo evaluate the possibility of retrieving leftover material of several viruses from a range of Ag-RDTs, for molecular genetic analysis.MethodsArchived Ag-RDTs which had been stored for up to 3 months at room temperature were used to extract viral nucleic acids for subsequent RT-qPCR, Sanger sequencing and Nanopore whole genome sequencing. The effects of brands of Ag-RDT and of various ways to prepare Ag-RDT material were evaluated.ResultsSARS-CoV-2 nucleic acids were successfully extracted and sequenced from nine different brands of Ag-RDTs for SARS-CoV-2, and for five of these, after storage for 3 months at room temperature. The approach also worked for Ag-RDTs for influenza virus (n = 3 brands), as well as for rotavirus and adenovirus 40/41 (n = 1 brand). The buffer of the Ag-RDT had an important influence on viral RNA yield from the test strip and the efficiency of subsequent sequencing.ConclusionOur finding that the test strip in Ag-RDTs is suited to preserve viral genomic material, even for several months at room temperature, and therefore can serve as source material for genetic characterisation could help improve global coverage of genomic surveillance for SARS-CoV-2 as well as for other viruses.

Despite the global use of rotavirus vaccines, vaccine breakthrough cases remain a pediatric health problem. In this study, we investigated suspected rotavirus vaccine breakthrough cases using next-generation sequencing (NGS)-based viral metagenomics ( n  = 102) and a panel of semiquantitative reverse transcription-PCR (RT-qPCR) ( n  = 92) targeting known enteric pathogens. Overall, we identified coinfections in 80% of the cases. Enteropathogens such as adenovirus (32%), enterovirus (15%), diarrheagenic Escherichia coli (1 to 14%), astrovirus (10%), Blastocystis spp. (10%), parechovirus (9%), norovirus (9%), Clostridioides (formerly Clostridium ) difficile (9%), Dientamoeba fragilis (9%), sapovirus (8%), Campylobacter jejuni (4%), and Giardia lamblia (4%) were detected. Except for a few reassortant rotavirus strains, unusual genotypes or genotype combinations were not present. However, in addition to well-known enteric viruses, divergent variants of enteroviruses and nonclassic astroviruses were identified using NGS. We estimated that in 31.5% of the patients, rotavirus was likely not the cause of gastroenteritis, and in 14.1% of the patients, it contributed together with another pathogen(s) to disease. The remaining 54.4% of the patients likely had a true vaccine breakthrough infection. The high prevalence of alternative enteropathogens in the suspected rotavirus vaccine breakthrough cases suggests that gastroenteritis is often the result of a coinfection and that rotavirus vaccine effectiveness might be underestimated in clinical and epidemiological studies.