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Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in‐house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV‐A71, echovirus 30, coxsackie A virus 21, and EV‐D68), HPeV3, and specificity controls. Reported results from 48 in‐house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In‐house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10−5). EV‐specific PCRs showed low cross‐reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in‐house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point‐of‐care tests. © 2019 The Authors. Journal of Medical Virology published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

ObjectiveTo test the hypothesis that enterovirus meningitis (EM) is a frequent and self-limiting condition, the epidemiology of EM in adults was examined.MethodsUsing 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).ResultsA 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.ConclusionsEM is common among young, healthy adults. Although the long-term prognosis remains reassuring, a substantial proportion have moderate disability at discharge, especially female patients.

Summary Background Enteroviruses can cause severe infections, especially in young children. Non-polio enterovirus infections are not notifiable in most countries in the EU and European Economic Area (EEA) region, and surveillance varies substantially between countries. We collected and analysed available enterovirus data across EU and EEA countries to assess the current epidemiological situation and need for standardising surveillance. Methods Aggregated data on any enterovirus detected between Jan 1, 2015, and Dec 31, 2017, through national enterovirus reference laboratories were requested from representatives in all 31 EU and EEA countries. Information collected included enterovirus types detected by month, patient age group, symptom, and specimen type. We also collected sequence data on viral capsid sequences for the three most clinically relevant enterovirus types, as identified from the data. Findings Aggregated data were provided by representatives from 24 (77%) of 31 countries. 9914 (66%) of 14 999 enterovirus infections with information about age were in children younger than 5 years, and 3197 (45%) of 7139 individuals for whom symptoms were reported had neurological symptoms. Other symptoms were non-specific fever (in 1607 [23%] patients), respiratory symptoms (in 1197 [17%] patients), hand, foot, and mouth disease (in 528 [7% patients), and myocarditis (in 39 [1%] patients). 68 deaths were temporally associated with enterovirus infection. Typing for 11 559 (67%) of 17 136 specimens revealed 66 enterovirus types. Coxsackievirus A6 was the most frequently detected enterovirus type (in 1556 [13%] of 11 559 typed enteroviruses), and 292 (65%) of 448 patients with coxsackievirus A6 infection with available clinical data presented with hand, foot, and mouth disease. Echovirus 30 was the second most frequently detected enterovirus type, representing 1412 (12%) of 11 559 typed enteroviruses, and 384 (82%) of 467 individuals with echovirus 30 infection with available clinical data had neurological symptoms. Sequences available from 18 countries showed circulation of newly emerging strains of enterovirus A71 and enterovirus D68. Interpretation To our knowledge, this study is the largest investigation of enterovirus circulation in EU and EEA countries and confirms the availability of non-polio enterovirus data in the region. Our study highlights the wide circulation of non-polio enteroviruses in Europe, mostly affecting young children and leading to neurological symptoms. Collecting data on morbidity and mortality related to enterovirus infections, as well as harmonising case definition for surveillance, should be encouraged. Funding None.

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.

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.

Objectives This study was performed to determine the seroprevalence and incidence of hepatitis E virus (HEV) infection among HIV-infected women during pregnancy and after delivery in a cohort of 200 Tanzanian women. Methods HIV-infected women participating in a study on antiretroviral therapy for the prevention of mother-to-child HIV transmission between 2006 and 2011, were tested retrospectively for anti-HEV immunoglobulin G (IgG) in plasma samples at 9 months post-partum. Anti-HEV IgG-positive patients were tested for anti-HEV IgG and immunoglobulin M (IgM) in samples from enrolment, and seroconverting women were tested for HEV RNA. Results A total of 16 women were anti-HEV IgG-positive, two of whom had seroconverted between enrolment and 9 months post-partum, with no detection of anti-HEV IgM or HEV RNA, yielding an HEV seroprevalence of 8.0% (confidence interval 5.0–12.6%) and an annual incidence rate of 1.0% (confidence interval 0.2–3.4%). CD4 cell counts were relatively high (median 403 × 106/l), with no significant difference between women with and without serological signs of HEV. Conclusions An annual HEV infection incidence rate of 1% strongly indicates ongoing transmission of HEV in Tanzania and should be kept in mind for pregnant women presenting with signs of acute hepatitis.

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

Picornaviruses (family Picornaviridae) are small, nonenveloped, positive-sense, single-stranded RNA viruses. The members of this family are currently classified into 47 genera and 110 species. Of picornaviruses, entero-and parechoviruses are associated with aseptic meningitis. They are transmitted via fecal-oral and respiratory routes, and occasionally, these viruses may cause a brief viremia and gain access to central nervous system (CNS). During the diagnostic screening of entero-and parechovirus types in Finland in year 2013-14, we detected a cluster of echovirus 4 (E4) infections in young adults and adolescents. As E4 is infrequently detected in Finland, we contacted several Northern and Central European laboratories that conduct routine surveillance for enteroviruses and, for those who have had E4 cases, we send a query for E4 sequences and data. Here we report CNS infections caused by E4 in Finland, Sweden, Norway, Denmark, Iceland and Germany in 2013 and 2014, and show that the E4 detected in these countries form a single lineage. In contrast, E4 strains circulating in these countries preceding the year 2013, and those circulating elsewhere in Europe during 2013-2014, formed several independent clusters.

Introduction Sequence-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. Aim The 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. Methods In 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. Results Of 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. Conclusions While HAV sequences in EU/EEA countries are comparable for surveillance, collaboration in sharing and comparing these can be further strengthened.

Background: Hepatitis E virus (HEV) genotype 3 is endemic in Europe, and the infection is mostly subclinical or acute and self-limiting. However, persistent infection is described among HIV-infected individuals. The prevalence of antibodies against HEV (anti-HEV) among HIV-infected persons varies geographically and is unknown in Denmark. Rates of co-infection with HEV among HIV-infected individuals in Denmark over three decades, from the early 1980s to 2013, were investigated. Methods: A total of 2506 HIV-infected persons were investigated from two cohorts followed at Hvidovre Hospital, Denmark. Blood samples were tested retrospectively for anti-HEV, including samples from 2216 persons who were enrolled in a prospective clinical cohort and followed between 1995 and 2013, as well as samples from 290 persons from a historical cohort followed between 1980 and 1994. For anti-HEV seroconverting individuals, serial samples were tested for HEV RNA. Factors associated with anti-HEV status were explored using multivariable logistic regression analysis. Results: The overall HEV seroprevalence rates were stable during the 1980s, 1990s, and 2000–2013 (23.1%, 22.9%, and 23.7%, respectively). In all decades, rates of anti-HEV increased with older age, and anti-HEV seropositivity was associated with older generations, HIV risk group, and geographic origin. Persistent HEV infection was not detected in any of 57 individuals with anti-HEV seroconversion. Conclusions: HEV seroprevalence rates were stable in HIV-infected individuals from the early 1980s to 2013. Rates increased with age. No evidence of persistent HEV infection was detected. Infection with HEV is frequent, but persistent HEV infection is rare among HIV-infected individuals. Keywords: Hepatitis E virus, HEV, Prevalence, HIV, Persistent HEV RNA

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205254/ 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. info:eu-repo/semantics/publishedVersion

SUMMARYRotavirus (RV) infections affect young children, but can also occur in adults. We sought to identify risk factors for RV infections in adults aged ⩾18 years in Denmark, and to describe illness and genotyping characteristics. From March 2005 to February 2009, we recruited consecutive cases of laboratory-confirmed RV infection and compared them with healthy controls matched by age, gender and municipality of residence. We collected information on illness characteristics and exposures using postal questionnaires. We calculated univariable and multivariable matched odds ratios (mOR) with conditional logistic regression. The study comprised 65 cases and 246 controls. Illness exceeded 10 days in 31% of cases; 22% were hospitalized. Cases were more likely than controls to suffer serious underlying health conditions [mOR 5·6, 95% confidence interval (CI) 1·7–18], and to report having had close contact with persons with gastrointestinal symptoms (mOR 9·4, 95% CI 3·6–24), in particular young children aged 18 years. Close contact with young children or adults with gastrointestinal symptoms is the main risk factor for RV infection in adults in Denmark. RV vaccination assessments should consider that RV vaccination in children may indirectly reduce the burden of disease in adults.

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. First, we thank the European Centre for Disease Prevention and Control (ECDC)’s Food- and Waterborne Diseases and Zoonoses team, particularly Ettore Severi and Johanna Takkinen for their support of this joint investigation. We also thank Lelia Thornton, HSE - Health Protection Surveillance Centre, Dublin, Ireland for sharing information on the Irish hepatitis A patient. Moreover, we greatly acknowledge the work of local and state health departments for their support of epidemiological investigations and molecular surveillance. Sí

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.

Background: The aim of this study was to investigate whether the syndrome New Neonatal Porcine Diarrhoea Syndrome (NNPDS) is associated with a viral aetiology. Four well-managed herds experiencing neonatal diarrhoea and suspected to be affected by NNPDS were included in a case-control set up. A total of 989 piglets were clinically examined on a daily basis. Samples from diarrhoeic and non-diarrhoeic piglets at the age of three to seven days were selected for extensive virological examination using specific real time polymerase chain reactions (qPCRs) and general virus detection methods. Results: A total of 91.7% of the animals tested positive by reverse transcription qPCR (RT-qPCR) for porcine kobuvirus 1 (PKV-1) while 9% and 3% were found to be positive for rotavirus A and porcine teschovirus (PTV), respectively. The overall prevalence of porcine astrovirus (PAstV) was 75% with 69.8% of the PAstV positive pigs infected with PAstV type 3. No animals tested positive for rotavirus C, coronavirus (TGEV, PEDV and PRCV), sapovirus, enterovirus, parechovirus, saffoldvirus, cosavirus, klassevirus or porcine circovirus type 2 (PCV2). Microarray analyses performed on a total of 18 animals were all negative, as were eight animals examined by Transmission Electron Microscopy (TEM). Using Next Generation de novo sequencing (de novo NGS) on pools of samples from case animals within all herds, PKV-1 was detected in four herds and rotavirus A, rotavirus C and PTV were detected in one herd each. Conclusions: Our detailed analyses of piglets from NNPDS-affected herds demonstrated that viruses did not pose a significant contribution to NNPDS. However, further investigations are needed to investigate if a systemic virus infection plays a role in the pathogenesis of NNPDS.

AIMS: To investigate seasonality and association of increased enterovirus and influenza activity in the community with ventricular fibrillation (VF) risk during first ST-elevation myocardial infarction (STEMI).METHODS: This study comprised all consecutive patients with first STEMI (n = 4,659; aged 18-80 years) admitted to the invasive catheterization laboratory between 2010-2016, at Copenhagen University Hospital, Rigshospitalet, covering eastern Denmark (2.6 million inhabitants, 45% of the Danish population). Hospital admission, prescription, and vital status data were assessed using Danish nationwide registries. We utilized monthly/weekly surveillance data for enterovirus and influenza from the Danish National Microbiology Database (2010-2016) that receives copies of laboratory tests from all Danish departments of clinical microbiology.RESULTS: Of the 4,659 consecutively enrolled STEMI patients, 581 (12%) had VF before primary percutaneous coronary intervention. In a subset (n = 807), we found that VF patients experienced more generalized fatigue and flu-like symptoms within 7 days before STEMI compared with the patients without VF (OR 3.39, 95% CI 1.76-6.54). During the study period, 2,704 individuals were diagnosed with enterovirus and 19,742 with influenza. No significant association between enterovirus and VF (OR 1.00, 95% CI 0.99-1.02), influenza and VF (OR 1.00, 95% CI 1.00-1.00), or week number and VF (p-value 0.94 for enterovirus and 0.89 for influenza) was found.CONCLUSION: We found no clear seasonality of VF during first STEMI. Even though VF patients had experienced more generalized fatigue and flu-like symptoms within 7 days before STEMI compared with patients without VF, no relationship was found between enterovirus or influenza exposure and occurrence of VF.

HPeV should be tested for in all young children suspected to have HPeV or enterovirus infection., 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.

Background The potential for outbreaks due to Enteroviruses (EV) with respiratory tropism, such as EV-D68, and the detection of new and rare EV species C is a concern. These EVs are typically not detected in stool specimens and may therefore be missed by standard EV surveillance systems. Following the North American outbreak of EV-D68 in 2014, Denmark piloted an enhanced EV surveillance system that included the screening of respiratory samples. Objectives We aim to report clinical manifestations and phylogenetic descriptions from the rare and emerging EVs identified thereby demonstrating the usefulness of this system. Study design Positive EV samples received through the enhanced non-polio EV pilot surveillance system were characterized by sequencing fragments of VP1, VP2 and VP4 capsid proteins and clinical observations were compiled. Results Between January 2015 and October 2016, six cases of rare genotypes EV-C104, C105 and C109 and nine cases of EV-D68 were identified. Patients presented with mild to moderately severe respiratory illness; no paralysis occurred. Distinct EV-C104, EV-C109 and EV-D68 sequences argue against a common source of introduction of these genotypes in the Danish population. Conclusions The enhanced EV surveillance system enabled detection and characterization of rare EVs in Denmark. In order to improve our knowledge of and our preparedness against emerging EVs, public health laboratories should consider expanding their EV surveillance system to include respiratory specimens.

Infections with human parechoviruses (HPeVs) are highly prevalent, particularly in neonates, where they may cause substantial morbidity and mortality. The clinical presentation of HPeV infection in neonates is indistinguishable from that of enterovirus (EV) infection and may vary from mild disease involving gastrointestinal and/or respiratory symptoms to more severe illness with sepsis-like symptoms such as fever, seizures, irritability, rash and/or feeding problems. HPeV is among the most common single causes of aseptic meningitis/meningoencephalitis in infants

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.

Coxsackievirus A6 (CV-A6) infection is a major etiologic agent for hand, foot and mouth disease (HFMD) in recent years. HFMD outbreaks associated with CV-A6 results from the evolutionary dynamics of CV-A6 and the appearance of novel recombinant forms (RFs). To examine this, 151 variants collected between 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 likelihood between CV-A6 genome recombination and sequence divergence (estimated substitution rate of 8.1 × 10-3 substitutions/site/year and RFs 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' untranslated regions. The emergence of new CV-A6 recombination groups has become widespread in Europe and Asia within the last 8 years.

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.

Summary Since its discovery in California in 1962, reports of enterovirus D68 have been infrequent. Before 2014, infections were confirmed in only 699 people worldwide. In August, 2014, two paediatric hospitals in the USA reported increases in the number of patients with severe respiratory illness, with an over-representation in children with asthma. Shortly after, the authorities recognised a nationwide outbreak, which then spread to Canada, Europe, and Asia. In 2014, more than 2000 cases of enterovirus D68 were reported in 20 countries. Concurrently, clusters of children with acute flaccid paralysis of unknown cause were reported in several US states and in Europe. Enterovirus D68 infection was confirmed in some of the paralysed children, but not all. Complications in patients who were severely neurologically affected resemble those caused by poliomyelitis. In this paper we systematically review reports on enterovirus D68 to estimate its global epidemiology and its ability to cause respiratory infections and neurological damage in children. We extracted data from 70 papers to report on prevalence, symptoms, hospitalisation and mortality, and complications of enterovirus D68, both before and during the large outbreak of 2014. The magnitude and severity of the enterovirus D68 outbreak underscores a need for improved diagnostic work-up of paediatric respiratory illness, not only to prevent unnecessary use of antibiotics, but also to ensure better surveillance of diseases. Existing surveillance systems should be assessed in terms of capacity and ability to detect and report any upsurge of respiratory viruses such as enterovirus D68 in a timely manner, and focus should be paid to development of preventive measures against these emerging enteroviruses that have potential for severe disease.

SUMMARYGroup A rotaviruses infect humans and a variety of animals. In July 2006 a rare rotavirus strain with G8P[14] specificity was identified in the stool samples of two adult patients with diarrheoa, who lived in the same geographical area in Denmark. Nucleotide sequences of the VP7, VP4, VP6, and NSP4 genes of the identified strains were identical. Phylogenetic analyses showed that both Danish G8P[14] strains clustered with rotaviruses of animal, mainly, bovine and caprine, origin. The high genetic relatedness to animal rotaviruses and the atypical epidemiological features suggest that these human G8P[14] strains were acquired through direct zoonotic transmission events.

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.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.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.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.

Background Sapoviruses are known to cause gastroenteritis mainly in young children. Objectives To establish a collection of sapoviruses and to gain knowledge about the genetic diversity and epidemiology of sapoviruses circulating in children in Denmark. Study design During a 24-month period in 2005–2007 samples from 1104 children, aged 0–3 years, submitted for acute gastroenteritis diagnostics, were analysed for sapoviruses by real-time PCR. Genotyping of positive findings was carried out by sequencing part of the capsid gene, and in several cases also part of the polymerase gene. Results Sapoviruses were detected in stool samples from 97 children (9%), with the highest prevalence in the 7–18 months age group. Viruses from three genogroups and seven genotypes were found. Genotype I.1 was demonstrated in half of the positive samples and was observed throughout the study period. The less common types seemed to appear during shorter periods, often in succession of each other. Positive samples were detected throughout the study period. The only months, in both years studied, with high proportions of positive samples were September, November and February. Conclusions Sapoviruses were commonly found in children with gastroenteritis in Denmark. No clear seasonal pattern could be seen. Genotype I.1 was clearly the most common genotype found, but several other genotypes circulated during shorter periods.

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.

One of the leading causes of severe childhood gastroenteritis are group A rotaviruses, and they have been found to be associated with similar to 40% of the annual gastroenteritis-associated hospitalizations in young Danish children One of the leading causes of severe childhood gastroenteritis are group A rotaviruses, and they have been found to be associated with ~40% of the annual gastroenteritis-associated hospitalizations in young Danish children

A food-borne outbreak of hepatitis A in Denmark was notified to other countries on 1 March 2013. A case-control study identified frozen berries eaten in smoothies as potential vehicle. In the following weeks, Finland, Norway and Sweden also identified an increased number of hepatitis A patients without travel history. Most cases reported having eaten frozen berries at the time of exposure. By 17 April, 71 cases were notified in the four countries. No specific type of berry, brand or origin of berries has yet been identified. .

Since November 2012, there has been an increase in reported cases of hepatitis A in tourists returning from Egypt in several European countries. As of 24 April, 80 HAV cases in travellers with symptom onset after 1 November 2012 visiting different areas in Egypt have been reported. Four cases from Norway, six cases from the Netherlands and five cases from England share an identical hepatitis A viral RNA sequence. This increase in cases suggests that vaccination recommendations for travellers to hepatitis A endemic countries should be reinforced.

Highlights • A novel enterovirus and parechovirus multiplex assay. • Extensive validation and implementation data. • Documentation of the assays performance during the first 15 months of routine testing., As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay. The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses in 171 (8%) and 66 (3%) of the samples, respectively. 180 of the positive samples could be genotyped by PCR and sequencing and the most common genotypes found were human parechovirus type 3, echovirus 9, enterovirus 71, Coxsackievirus A16, and echovirus 25. During 2009 in Denmark, both enterovirus and human parechovirus type 3 had a similar seasonal pattern with a peak during the summer and autumn. Human parechovirus type 3 was almost invariably found in children less than 4 months of age. In conclusion, a multiplex assay was developed allowing simultaneous detection of 2 viruses, which can cause similar clinical symptoms.

Group A rotaviruses can infect both humans and animals. Individual rotavirus strains can occasionally cross species barriers and might hereby contribute to the emergence of new genotypes in heterologous hosts. The incidence and impact of zoonotic rotavirus are not well defined, and one reason for this is a lack of data about strains circulating in suspected reservoir animal hosts. In this study we report the incidence, genetic diversity, and molecular epidemiology of rotaviruses detected in domestic cattle and swine in 6 European countries. From 2003 to 2007, 1101 and more than 2000 faecal specimens were collected from swine and cattle, both healthy and diarrhoeic, and tested for rotaviruses. Viruses from positive stools were genotyped and a subset of strains was characterized by nucleotide sequencing and phylogenetic analysis of the VP7 (G) and VP4 (P) genes. Rotaviruses were detected in 43% of bovine samples and in 14% of porcine samples. In cattle, 10 different combinations of G and P types were identified and the most common strains were G6P[11] and G6P[5]. In swine, the number of identified G-P combinations was higher (n=21), however, no single combination was predominant across Europe. Newly described genotype specificities, P[27] and P[32], were identified in swine. When compared at the nucleotide sequence level, the identified porcine rotavirus strains and contemporary human strains grouped together phylogenetically, whereas bovine rotavirus strains formed separate clades. These data demonstrate large genetic diversity of porcine and bovine rotavirus strains across Europe, and suggest that livestock herds may serve as potential reservoirs for human infections.

International audience; BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was

Hepatitis B virus (HBV) infection is widely distributed in both human and ape populations throughout the world and is a major cause of human morbidity and mortality. HBV variants are currently classified into the human genotypes A to H and species-associated chimpanzee and gibbon/orangutan groups. To examine the role of recombination in the evolution of HBV, large-scale data retrieval and automated phylogenetic analysis (TreeOrder scanning) were carried out on all available published complete genome sequences of HBV. We detected a total of 24 phylogenetically independent potential recombinants (different genotype combinations or distinct breakpoints), eight of which were previously undescribed. Instances of intergenotype recombination were observed in all human and ape HBV variants, including evidence for a novel gibbon/genotype C recombinant among HBV variants from Vietnam. By recording sequence positions in trees generated from sequential fragments across the genome, violations of phylogeny between trees also provided evidence for frequent intragenotype recombination between members of genotypes A, D, F/H, and gibbon variants but not in B, C, or the Asian B/C recombinant group. In many cases, favored positions for both inter- and intragenotype recombination matched positions of phylogenetic reorganization between the human and ape genotypes, such as the end of the surface gene and the core gene, where sequence relationships between genotypes changed in the TreeOrder scan. These findings provide evidence for the occurrence of past, extensive recombination events in the evolutionary history of the currently classified genotypes of HBV and potentially in changes in its global epidemiology and associations with human disease.

Acute flaccid myelitis; Enterovirus D68; Surveillance Mielitis flàcida aguda; Enterovirus D68; Vigilància Mielitis flácida aguda; Enterovirus D68; Vigilancia 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.

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. ispartof: Journal of Clinical Virology vol:101 pages:11-17 ispartof: location:Netherlands status: published

From June 2014 through February 2015, respiratory samples from 130 Danish patients were screened for enterovirus D68 (EV-D68). Fourteen EV-D68 cases were detected, of which 12 presented with respiratory symptoms, and eight had known underlying disease. The median age of EV-D68 cases was three years (interquartile range: 0–30 years). Acute flaccid paralysis (AFP) was not detected although Danish EV-D68 strains showed > 98% nt identity with EV-D68-strains from AFP cases from the United States and France.

Enterovirus (EV) 71 has emerged as a primary cause of severe neurologic enterovirus infection in the aftermath of the global polio eradication effort. Eleven subgenotypes of EV71 exist, the C4 subgenotype being associated with large outbreaks in Asia with high mortality rates. This subgenotype has rarely been reported in Europe. In the period between 1 January 2009 and 31 December 2013 a total of 1,447 EV positive samples from 1,143 individuals were sent to the Statens Serum Institute (SSI), and 938 samples from 913 patients were genotyped at the Danish National World Health Organization Reference laboratory for Poliovirus at SSI. Echovirus 6 (E06) (n=141 patients), echovirus 30 (E30) (n=114), coxsackievirus A6 (CA06) (n=96) and EV71 (n=63) were the most prevalent genotypes. We observed a shift in circulating EV71 subgenotypes during the study period, with subgenotype C4 dominating in 2012. A total of 34 EV71 patients were found to be infected with strains of the C4 subgenotype, and phylogenetic analysis revealed that they belonged to the C4a lineage. In our study, the proportions of cases with cerebral and/or sepsis-like symptoms were similar in those affected by C4a (19/34) and those with C1 and C2 (15/35). The majority (n=30) of the 34 EV71 C4 cases were children ≤5 years of age, and males (n=22) were over-represented. Continued EV surveillance is required to monitor the spread of EV71 C4 in Denmark and the rest of Europe.