Detecting SARS-CoV-2 Omicron B.1.1.529 Variant in Wastewater Samples by Using Nanopore Sequencing.

We report wastewater surveillance for SARS-CoV-2 variants of concern by using mutation-specific, real-time PCR and rapid nanopore sequencing. This surveillance might be useful for an early warning in a scenario in which a new variant is emerging, even in areas that have low virus incidences.

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.

Long-Term Survival, Health, Social Functioning, and Education in Patients With an Enterovirus Central Nervous System Infection, Denmark, 1997-2016.

BACKGROUND: The long-term clinical course of patients with an enterovirus central nervous system infection (ECI) is poorly understood. METHODS: We performed a nationwide population-based cohort study of all Danish patients with ECI diagnosed 1997-2016 (n = 1745) and a comparison cohort from the general population individually matched on date of birth and sex (n = 17 450). Outcomes were categorized into mortality and risk of cancer and likely measures of neurological sequelae: neuropsychiatric morbidities, educational landmarks, use of hospital services, employment, receipt of disability pension, income, number of sick leave days, and nursing home residency. RESULTS: Mortality in the first year was higher among patients with ECI (mortality rate ratio [MRR] = 10.0; 95% confidence interval [CI], 4.17-24.1), but thereafter mortality was not higher (MMR = 0.94; 95% CI, 0.47-1.86). Long-term outcomes for patients with ECI were not inferior to those of the comparison cohort for risk of cancer, epilepsy, mental and behavioral disorders, dementia, depression, school start, school marks, high school education, use of hospital services, employment, receipt of disability pension, income, days of sick leave, or nursing home residency. CONCLUSIONS: Diagnosis of an ECI had no substantial impact on long-term survival, health, or social/educational functioning.

Co-circulation of multiple enterovirus D68 subclades, including a novel B3 cluster, across Europe in a season of expected low prevalence, 2019/20.

Enterovirus D68 (EV-D68) was detected in 93 patients from five European countries between 1 January 2019 and 15 January 2020, a season with expected low circulation. Patients were primarily children (n = 67, median age: 4 years), 59 patients required hospitalisation and five had severe neurologic manifestations. Phylogenetic analysis revealed two clusters in the B3 subclade and subclade A2/D. This circulation of EV-D68 associated with neurological manifestations stresses the importance of surveillance and diagnostics beyond expected peak years.

Improving preparedness to respond to cross-border hepatitis A outbreaks in the European Union/European Economic Area: towards comparable sequencing of hepatitis A virus.

IntroductionSequence-based typing of hepatitis A virus (HAV) is important for outbreak detection, investigation and surveillance. In 2013, sequencing was central to resolving a large European Union (EU)-wide outbreak related to frozen berries. However, as the sequenced HAV genome regions were only partly comparable between countries, results were not always conclusive.AimThe objective was to gather information on HAV surveillance and sequencing in EU/European Economic Area (EEA) countries to find ways to harmonise their procedures, for improvement of cross-border outbreak responses.MethodsIn 2014, the European Centre for Disease Prevention and Control (ECDC) conducted a survey on HAV surveillance practices in EU/EEA countries. The survey enquired whether a referral system for confirming primary diagnostics of hepatitis A existed as well as a central collection/storage of hepatitis A cases' samples for typing. Questions on HAV sequencing procedures were also asked. Based on the results, an expert consultation proposed harmonised procedures for cross-border outbreak response, in particular regarding sequencing. In 2016, a follow-up survey assessed uptake of suggested methods.ResultsOf 31 EU/EEA countries, 23 (2014) and 27 (2016) participated. Numbers of countries with central collection and storage of HAV positive samples and of those performing sequencing increased from 12 to 15 and 12 to 14 respectively in 2016, with all countries typing an overlapping fragment of 218 nt. However, variation existed in the sequenced genomic regions and their lengths.ConclusionsWhile HAV sequences in EU/EEA countries are comparable for surveillance, collaboration in sharing and comparing these can be further strengthened.

Transfusion transmission of hepatitis A virus with fecal shedding in a previously hepatitis A vaccinated recipient.

We describe a rare case of hepatitis A virus (HAV) replication in feces despite presence of hepatitis A antibodies in an acute myeloid leukemia (AML) patient after transfusion with HAV contaminated platelets. The patient has been vaccinated against HAV years before the AML diagnosis. Transient infection and reshedding should thus be considered in antibody-positive hematological patients. Transfusion associated HAV transmission is rare, and little evidence exists on the clinical consequences and possible effect of treatment with immunoglobulin. Further reporting on fecal shedding despite antibodies are needed, as HAV antibody levels are used as course of action for post-exposure prophylaxis and infection control.

Two concurrent outbreaks of hepatitis A highlight the risk of infection for non-immune travellers to Morocco, January to June 2018.

From January to June 2018, two ongoing hepatitis A outbreaks affected travellers returning from Morocco and cases in Europe without travel history, resulting in 163 patients in eight European countries. Most interviewed travel-related cases were unaware of the hepatitis A risk in Morocco. Molecular analysis revealed two distinct hepatitis A virus (HAV) strains (subgenotype IA DK2018_231; subgenotype IB V18-16428). Vaccination recommendations should be emphasised to increase awareness among non-immune travellers to Morocco and HAV-endemic countries.

Hepatitis A outbreak disproportionately affecting men who have sex with men (MSM) in the European Union and European Economic Area, June 2016 to May 2017.

Between 1 June 2016 and 31 May 2017, 17 European Union (EU) and European Economic Area countries reported 4,096 cases associated with a multi-country hepatitis A (HA) outbreak. Molecular analysis identified three co-circulating hepatitis A virus (HAV) strains of genotype IA: VRD_521_2016, V16-25801 and RIVM-HAV16-090. We categorised cases as confirmed, probable or possible, according to the EU outbreak case definitions. Confirmed cases were infected with one of the three outbreak strains. We investigated case characteristics and strain-specific risk factors for transmission. A total of 1,400 (34%) cases were confirmed; VRD_521_2016 and RIVM-HAV16-090 accounted for 92% of these. Among confirmed cases with available epidemiological data, 92% (361/393) were unvaccinated, 43% (83/195) travelled to Spain during the incubation period and 84% (565/676) identified as men who have sex with men (MSM). Results depict an HA outbreak of multiple HAV strains, within a cross-European population, that was particularly driven by transmission between non-immune MSM engaging in high-risk sexual behaviour. The most effective preventive measure to curb this outbreak is HAV vaccination of MSM, supplemented by primary prevention campaigns that target the MSM population and promote protective sexual behaviour.

Co-circulation of multiple subtypes of enterovirus A71 (EV- A71) genotype C, including novel recombinants characterised by use of whole genome sequencing (WGS), Denmark 2016.

In Europe, enterovirus A71 (EV-A71) has primarily been associated with sporadic cases of neurological disease. The recent emergence of new genotypes and larger outbreaks with severely ill patients demonstrates a potential for the spread of new, highly pathogenic EV-A71 strains. Detection and characterisation of these new emerging EV variants is challenging as standard EV assays may not be adequate, necessitating the use of whole genome analysis.

Severe Human Parechovirus Infections in Infants and the Role of Older Siblings.

Human parechovirus (HPeV) is a cause of severe morbidity among infants and young children. To evaluate the associations between early environmental risk factors and HPeV infections, we carried out a nationwide cohort study linking registry data on birth and sibship characteristics with a laboratory surveillance database, covering all HPeV infections detected in Denmark during 2009-2012 among children <5 years of age. Incidence rate ratios were calculated in log-linear Poisson regression analyses. Overall, 133 HPeV infections, 85 caused by human parechovirus type 3 (HPeV-3) and 48 by human parechovirus other than type 3 (non-HPeV-3), were detected among 132 children. Neither birth weight, mode of delivery, Apgar score, nor gestational age was associated with the risk of HPeV infections. Compared with firstborn children, secondborn children were at a 9-fold increased risk (incidence rate ratio = 8.68, 95% confidence interval: 3.85, 19.53) of contracting HPeV-3 infections, but at no increased risk of contracting non-HPeV-3 infections. However, the shorter the age gap to the nearest older sibling, the higher the risk of HPeV-3 as well as non-HPeV-3 infections, although the trend was strongest for HPeV-3 infections. Our study is the first to suggest that having a slightly older sibling increases the risk for severe neonatal HPeV infections. This new knowledge might lead to new preventive measures.

Molecular epidemiology and the evolution of human coxsackievirus A6.

Coxsackievirus A6 (CV-A6) is a major aetiologic agent for hand, foot and mouth disease (HFMD) in recent years. HFMD outbreaks associated with CV-A6 resulted from the evolutionary dynamics of CV-A6 and the appearance of novel recombinant forms (RFs). To examine this, 151 variants collected in 2013 and 2014 from Germany, Spain, Sweden, Denmark and Thailand were genotyped for the VP1 capsid and 3Dpol genes. Analysis of the VP1 gene showed an increasing correspondence between CV-A6 genome recombination and sequence divergence (estimated substitution rate of 8.1×10-3 substitutions site-1 year-1 and RF half-life of 3.1 years). Bayesian phylogenetic analysis showed that recent recombination groups (RF-E, -F, -H, -J and -K) shared a common ancestor (RF-A). Thirty-nine full-length genomes of different RFs revealed recombination breakpoints between the 2A-2C and the 5' UTRs. The emergence of new CV-A6 recombination groups has become widespread in Europe and Asia within the last 8 years.

Low transfusion transmission of hepatitis E among 25,637 single-donation, nucleic acid-tested blood donors.

BACKGROUND: Hepatitis E virus genotype-3 (HEV-gt-3) causes autochthonous infections in western countries, with a primary reservoir in animals, especially pigs. HEV transfusion transmission has been reported, and HEV-gt-3 prevalence is high in some European countries. The prevalence of HEV RNA was investigated among Danish blood donors, and the prevalence of HEV transfusion-transmitted infection (TTI) was investigated among recipients. STUDY DESIGN AND METHODS: Samples from 25,637 consenting donors collected during 1 month in 2015 were screened retrospectively using an individual-donation HEV RNA nucleic acid test with a 95% detection probability of 7.9 IU/mL. HEV-positive samples were quantified by real-time polymerase chain reaction and genotyped. Transmission was evaluated among recipients of HEV RNA-positive blood components. Phylogenetic analyses compared HEV sequences from blood donors, symptomatic patients, and swine. RESULTS: Eleven donations (0.04%) were confirmed as positive for HEV RNA (median HEV RNA level, 13 IU/mL). Two donations were successfully genotyped as HEV-gt-3. Only one donor had a travel history outside Europe. Nine of 11 donors were male, but the gender ratio was nonsignificant compared with the total donor population. Seven available recipients tested negative for HEV RNA and anti-HEV immunoglobulin M in follow-up samples. One recipient was HEV RNA-negative but anti-HEV immunoglobulin G-positive. HEV TTI was considered unlikely, but a transfusion-induced secondary immune response could not be excluded. Phylogenetic analysis showed relatively large sequence differences between HEV from donors, symptomatic patients, and swine. CONCLUSIONS: Despite an HEV RNA prevalence of 0.04% in Danish blood donations, all HEV-positive donations carried low viral loads, and no evidence of TTI was found.

Evaluation of the enterovirus laboratory surveillance system in Denmark, 2010 to 2013.

The primary aim of the Danish enterovirus (EV) surveillance system is to document absence of poliovirus infection. The conflict in Syria has left many children unvaccinated and movement from areas with polio cases to Europe calls for increased awareness to detect and respond to virus-transmission in a timely manner. We evaluate the national EV laboratory surveillance, to generate recommendations for system strengthening. The system was analysed for completeness of viral typing analysis and clinical information and timeliness of specimen collection, laboratory results and reporting of clinical information. Of 23,720 specimens screened, 2,202 (9.3%) were EV-positive. Submission of cerebrospinal fluid and faecal specimens from primary diagnostic laboratories was 79.5% complete (845/1,063), and varied by laboratory and patient age. EV genotypes were determined in 68.5% (979/1,430) of laboratory-confirmed cases, clinical information was available for 63.1% (903/1,430). Primary diagnostic results were available after a median of 1.4 days, typing results after 17 days, detailed clinical information after 33 days. The large number of samples typed demonstrated continued monitoring of EV-circulation in Denmark. The system could be strengthened by increasing the collection of supplementary faecal specimens, improving communication with primary diagnostic laboratories, adapting the laboratory typing methodology and collecting clinical information with electronic forms.

Human parechovirus infection, Denmark.

Human parechoviruses (HPeVs) often cause severe illness among young children. National surveillance with routine testing of all cerebrospinal fluid, fecal, and tissue samples was conducted during January 2009-December 2012 in all counties in Denmark (6,817 samples from 4,804 children were screened for HPeV). We detected HPeV RNA in 202 (3.0%) specimens from 149 persons. Young infants were at highest risk for HPeV, and 9 (6%) of the HPeV-infected children died, probably of their HPeV illness. HPeV3 was the most common genotype identified, and 5 closely related clades of HPeV3 circulated in Denmark throughout the study period. Our study adds perspective on the prevalence and clinical and molecular virologic characteristics of HPeV infection.

Results from the SARS-CoV-2 wastewater-based surveillance system in Denmark, July 2021 to June 2022.

The microbiological analysis of wastewater samples is increasingly used for the surveillance of SARS-CoV-2 globally. We described the setup process of the national SARS-CoV-2 wastewater-based surveillance system in Denmark, presented its main results during the first year of activities, from July 2021 to June 2022, and discussed their operational significance. The Danish SARS-CoV-2 wastewater-based surveillance system was designed to cover 85 % of the population in Denmark and it entailed taking three weekly samples from 230 sites. Samples were RT-qPCR tested for SARS-CoV-2 RNA, targeting the genetic markers N1, N2 and RdRp, and for two faecal indicators, Pepper Mild Mottle Virus and crAssphage. We calculated the weekly SARS-CoV-2 RNA concentration in the wastewater from each sampling site and monitored it in view of the results from individual testing, at the national and regional levels. We attempted to use wastewater results to identify potential local outbreaks, and we sequenced positive wastewater samples using Nanopore sequencing to monitor the circulation of viral variants in Denmark. The system reached its full implementation by October 2021 and covered up to 86.4 % of the Danish population. The system allowed for monitoring of the national and regional trends of SARS-CoV-2 infections in Denmark. However, the system contribution to the identification of potential local outbreaks was limited by the extensive information available from clinical testing. The sequencing of wastewater samples identified relevant variants of concern, in line with results from sequencing of human samples. Amidst the COVID-19 pandemic, Denmark implemented a nationwide SARS-CoV-2 wastewater-based surveillance system that integrated routine surveillance from individual testing. Today, while testing for COVID-19 at the community level has been discontinued, the system is on the frontline to monitor the occurrence and spread of SARS-CoV-2 in Denmark.

Acute flaccid rhombencephalomyelitis with radiculitis in a child with an enterovirus A71 infection seen for the first time in Denmark: a case report.

BACKGROUND: Acute flaccid myelitis is a serious condition of the spinal cord. More than 80% of patients experience a mild respiratory illness or fever consistent with a viral infection prior to acute flaccid myelitis development. Enterovirus A71 is known to circulate in Denmark, and has previously been associated with severe neurological symptoms. In this case report we describe acute flaccid rhombencephalomyelitis with radiculitis in an infant with an enterovirus infection. CASE PRESENTATION: The 8-month-old male of Asian origin presented with fever and gastrointestinal symptoms, followed by severe neurological deficits such as flaccid paralysis of the neck and upper extremities. An initial magnetic resonance imaging scan of the brain was normal, and the boy was treated for encephalitis. A follow-up magnetic resonance imaging scan of the brain and spinal cord 1 week later showed the development of pathological symmetrical gray matter hyperintensity lesions on T2-weighted images in the brainstem and upper medulla spinalis, and nerve enhancement in the terminal thread of the spinal cord and the cervical roots; findings consistent with rhombencephalomyelitis with radiculitis causing flaccid paralysis. Enterovirus A71 was detected in both nasopharyngeal and fecal specimens. Other differential diagnostic etiologies of viral and bacterial encephalitis, including poliovirus, were excluded. CONCLUSIONS: This is the first case in Denmark of a patient diagnosed with acute flaccid rhombencephalomyelitis strongly linked to an enterovirus A71 infection. This case emphasizes the diagnostic importance of combining a history of respiratory and/or gastrointestinal illness, fever, and delayed onset of varying degrees of paralysis with progressive characteristic spinal and brain lesions. Analysis of respiratory, fecal, and cerebrospinal samples for the presence of enterovirus, and eliminating other differential pathogens, is essential to confirm the diagnosis.

Clinical and virological features of enterovirus 71 infections in Denmark, 2005 to 2008.

BACKGROUND: Since the late 1990s enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth disease with fatal cases especially in the Asian Pacific region. The objective of this study was to describe the clinical and virological features of EV71 infections in Denmark. METHODS: All enterovirus-positive samples in Denmark are submitted to the National Poliovirus Laboratory for typing, and the EV71-positive samples are characterized by sequencing and phylogenetic analysis. Clinical information was gathered for the EV71-positive patients. RESULTS: During 2005-2008, EV71 was demonstrated in 29 patients. In 2007 EV71 was the second most common enterovirus type detected in Denmark. Twenty-one of the 29 patients were children aged ≤1 y, 24 were hospitalized, and meningitis was the most common diagnosis. Gastroenteritis and hand, foot and mouth disease were other common clinical manifestations, but no fatal cases or cases of pulmonary oedema were seen. A novel subgenotype in Europe, B5, dominated the 2007 outbreak, but co-circulated with subgenotypes C1 and C2. CONCLUSIONS: In conclusion EV71 was among the common enterovirus types in Denmark, and in 2007 a novel subgenotype, B5, was observed. EV71 was mainly diagnosed in infants, and the majority of patients were hospitalized with meningitis.

Enterovirus Meningitis in Adults: A Prospective Nationwide Population-Based Cohort Study.

OBJECTIVE: To test the hypothesis that enterovirus meningitis (EM) is a frequent and self-limiting condition, the epidemiology of EM in adults was examined. METHODS: Using a prospective, nationwide, population-based database, all adults with EM confirmed by PCR of the CSF from 2015 to 2019 were included. Unfavorable outcome was defined as Glasgow Outcome Scale scores of 1-4 at discharge. Modified Poisson regression was used to compute adjusted relative risks (RRs). RESULTS: A total of 419 cases of EM in 418 adults (46% female, median age 31 years [interquartile range (IQR) 27-35]) yielded an incidence of 1.80/100,000/year. Admission diagnoses included CNS infection 247/397 (62%), other neurologic conditions 89/397 (22%), and cerebrovascular diseases 33/397 (8%). Genotype was available for 271 cases, of which echovirus 30 accounted for 155 (57%). Patients presented with headache 412/415 (99%), history of fever 303/372 (81%), photophobia 292/379 (77%), and neck stiffness 159/407 (39%). Fever (≥38.0°C) was observed in 192/399 (48%) at admission. The median CSF leukocyte count was 130 106/L (range 0-2,100) with polymorphonuclear predominance (>50%) in 110/396 (28%). Cranial imaging preceded lumbar puncture in 127/417 (30%) and was associated with non-CNS infection admission diagnoses and delayed lumbar puncture (median 4.8 hours [IQR 3.4-7.9] vs 1.5 [IQR 0.8-2.8], p < 0.001). Unfavorable outcome occurred in 99/419 (24%) at discharge; more often in female patients (RR 2.30 [1.58-3.33]) and less frequent in echovirus 30 (RR 0.67 [0.46-1.00]) in adjusted analyses. Outcome remained unfavorable in 22/379 (6%) after 6 months. CONCLUSIONS: EM is common among young, healthy adults. Although the long-term prognosis remains reassuring, a substantial proportion have moderate disability at discharge, especially female patients.