Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021-2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study.

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.

Molecular epidemiology and characterization of enteroviruses detected in cerebrospinal fluid and respiratory samples in Slovenia, 2014-2023.

Enterovirus (EV) infections have various symptoms and severe complications, including death. To determine EV prevalence and EV types in Slovenia, data on over 25 000 EV RNA tests for diagnostics and surveillance from 2014 to 2023 were analyzed. Altogether, 3733 cerebrospinal fluid (CSF) and 21 297 respiratory (sentinel and clinical) samples were tested for EV RNA. EV typing was performed on all residual EV-positive CSF samples and on subset of respiratory specimens. Altogether, 1238 samples tested positive for EV RNA: 238 (6.4%) CSF and 1000 (4.7%) respiratory samples. EV-positive patients were predominantly male (p < 0.001). Many EV-positive CSF samples were from infants under 3 months (33.1%), whereas most EV-positive respiratory samples were from children 1 to 2 years old (49.2%). Echovirus 30 (E-30) was most frequent in CSF (33.0%), followed by CV-B5 (13.8%) and E-6 (13.8%). CV-A6 was most frequent in respiratory samples (16.0%), followed by EV-D68 (7.6%) and CV-A5 (7.4%). EV types in CSF and respiratory samples show diverse dynamics, with some outbreaks indicated. A significant difference was found in the EV detection rate between CSF and respiratory samples by age. Various EV types were characterized, showing that some EV types are more neurotropic or cause more severe infections.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Delayed Laboratory Response to COVID-19 Caused by Molecular Diagnostic Contamination.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries.

Seasonality and Genotype Diversity of Human Rhinoviruses during an Eight-Year Period in Slovenia.

Due to the high socioeconomic burden of rhinoviruses, the development of prevention and treatment strategies is of high importance. Understanding the epidemiological and clinical features of rhinoviruses is essential in order to address these issues. Our study aimed to define the seasonality and molecular epidemiology of rhinoviruses in Slovenia. Over a period of eight years, a total of 20,425 patients from sentinel primary healthcare settings and sentinel hospitals were examined for a panel of respiratory viruses in the national programme for the surveillance of influenza-like illnesses and acute respiratory infections. The patients were from all age groups and had respiratory infections of various severity. Infection with a rhinovirus was confirmed using an RT-rPCR in 1834 patients, and 1480 rhinoviruses were genotyped. The molecular analysis was linked to demographical and meteorological data. We confirmed the year-round circulation of rhinoviruses with clear seasonal cycles, resulting in two seasonal waves with peaks in spring and autumn. High levels of genotype variability and co-circulation were confirmed between and within seasons and were analysed in terms of patient age, the patient source reflecting disease severity, and meteorological factors. Our study provides missing scientific information on the genotype diversity of rhinoviruses in Slovenia. As most previous investigations focused on exclusive segments of the population, such as children or hospitalised patients, and for shorter study periods, our study, with its design, size and length, contributes complementary aspects and new evidence-based knowledge to the regional and global understanding of rhinovirus seasonality and molecular epidemiology.

Detection of SARS-CoV-2 RNA in hospital wastewater from a low COVID-19 disease prevalence area.

Previous studies on SARS-CoV and MERS-CoV reported the detection of viral RNA in the stool of both symptomatic and asymptomatic individuals. These clinical observations suggest that municipal and hospital wastewater from affected communities may contain SARS-CoV-2 RNA. Recent studies have also reported the presence of SARS-CoV-2 RNA in human feces. Wastewater-based epidemiology (WBE) is a promising approach to understand the prevalence of viruses in a given catchment population, as wastewater contains viruses from symptomatic and asymptomatic individuals. The current study reports the first detection of SARS-CoV-2 RNA in untreated wastewater in Slovenia. Two sizes of centrifugal filters were tested: 30 kDa and 10 kDA AMICON® Ultra-15 Centrifugal Filters, where 10 kDA resulted in a higher concentration factor and higher recovery efficiency. The results in hospital wastewater show that WBE can be used for monitoring COVID -19 and could be applied in municipal wastewater treatment plants as a potential complementary tool for public health monitoring at population level.

European Non-Polio Enterovirus Network: Introduction of Hospital-Based Surveillance Network to Understand the True Disease Burden of Non-Polio Enterovirus and Parechovirus Infections in Europe.

BACKGROUND: Non-polio enteroviruses (EVs) and human parechoviruses (PeVs) cause a wide range of human infections. Limited data on their true disease burden exist as standardized European-wide surveillance is lacking. AIMS: Our aim is to estimate the disease burden of EV and PeV infections in Europe via establishment of standardized surveillance for hand, foot and mouth disease (HFMD) and respiratory and neurological infections caused by these viruses. We will also assess the sensitivity of assays implemented in the network of participating laboratories so that all EV and PeV types are adequately detected. Plan. The European Non-Polio Enterovirus Network (ENPEN) has developed standardized protocols for a prospective, multi-center and cross-sectional hospital-based pilot study. Protocols include guidance for diagnosis, case definition, detection, characterization and reporting of EV and PeV infections associated with HFMD and respiratory and neurological diseases. Over 30 sites from 17 European countries have already registered to this one pilot study, likely to be commenced in 2022. BENEFITS: This surveillance will allow European-wide comparison of data on EV and PeV infection. These data will also be used to determine the burden of EV and PeV infections, which is needed to guide the further prevention measures and policies.

Varicella susceptibility and transmission dynamics in Slovenia.

BACKGROUND: A cross-sectional, age-stratified study was conducted to determine varicella-zoster seroprevalence and force of infection in Slovenia. METHODS: 3689 serum samples were tested for VZV IgG antibodies with an enzyme immunoassay. Semiparametric and parametric modelling were used to estimate the force of infection. RESULTS: Overall, 85.6% of serum samples were seropositive. Age-specific prevalence rose rapidly in preschool children and over 90% of 8 years old tested positive for VZV. However, 2.8% of serum samples among women of childbearing age were seronegative. Semiparametric modelling yielded force of infection estimates of 0.182 (95% CI 0.158-0.206), 0.367 (95% CI 0.285-0.448) and 0.008 (95% CI 0.0-0.032) for age groups 0.5- < 6, 6-11 and >or=12 years, respectively, and 0.175 (95% CI 0.147-0.202), 0.391 (95% CI 0.303-0.480) and 0.025 (95% CI 0.003-0.046) for age groups 0.5- < 5, 5-9 and >or=10 years, respectively. CONCLUSIONS: Regardless of the age grouping used, the highest transmission occurred in children in their first years of school.

High seroprevalence of varicella, measles, mumps, rubella and pertussis antibodies in first-grade medical students.

OBJECTIVE: The aim of the study was to investigate seropositivity for five vaccine-preventable communicable diseases - varicella, measles, mumps, rubella and pertussis - in a sample of first-grade medical students. METHODS: A total of 256 students were tested for varicella IgG antibodies. In addition, 138 of the students were serologically screened for measles, mumps, rubella and pertussis antibodies. Data on vaccination and history of measles, mumps, rubella and pertussis were collected. RESULTS: Immunity to varicella, measles, mumps and rubella was established in 97.6%, 96.4%, 97.8% and 99.3% participants, respectively. Anti-pertussis toxin antibodies were detected in 81.2% of the students. CONCLUSIONS: The rates of varicella, measles, mumps and rubella immunity in first-grade medical students are very high. To identify those students who need vaccination before commencing practical work in healthcare facilities, a meticulous disease and vaccination history should be taken and medical records should be reviewed. Medical students providing no written evidence of adequate vaccination status should be serologically tested and vaccinated if necessary.

Recommendations for enterovirus diagnostics and characterisation within and beyond Europe.

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5'noncoding regions (5'NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5'NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden.

A comparison of the demographic and clinical characteristics of laboratory-confirmed influenza B Yamagata and Victoria lineage infection.

BACKGROUND: The evolution of influenza viruses is characterized by the co-circulation of two antigenically and genetically distinct lineages B/Victoria/2/87-like viruses (Victoria) and B/Yamagata/16/88-like viruses (Yamagata). To date, there is not much data associating lineages variation with demographic and clinical data. OBJECTIVES: We compared the demographic and clinical characteristics of patients with laboratory-confirmed influenza B Victoria or Yamagata lineage infection. STUDY DESIGN: We retrospectively analyzed data from 6811 patients aged from <1 through 99 years with influenza like-illness who consulted the sentinel site or sentinel hospital in the seasons 2010/2011, 2011/2012 and 2012/2013. There were 662 patients positive for influenza B virus by multiplex real-time RT-PCR. Six hundred thirty-seven (96.8%) were successfully subtyped for Victoria or Yamagata lineage infection. The available demographic and clinical data was compared. RESULTS: Patients with the Victoria lineage were significantly younger compared to patients infected with the Yamagata lineage. The Victoria lineage was the predominant strain in the 15-34 age groups in patients consulting at primary care level and in hospitalized patients. In the youngest age group (0-4 years) approximately half of the confirmed influenza B cases belonged to the Victoria (55%) and the rest to the Yamagata lineage (45%). Aside from age, there was no statistically significant difference found in gender distribution, vaccination history, clinical presentation or risk factors for severe influenza infection in hospitalized patients after adjustment for the age. CONCLUSIONS: The frequency of influenza B Victoria and Yamagata infection is age dependent with no significant differences detected in clinical presentation comparing both lineages.