Functional SARS-CoV-2 cross-reactive CD4+ T cells established in early childhood decline with age.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (ß-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein-Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination.

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.

Evolution, geographic spreading, and demographic distribution of Enterovirus D68.

Worldwide outbreaks of enterovirus D68 (EV-D68) in 2014 and 2016 have caused serious respiratory and neurological disease. We collected samples from several European countries during the 2018 outbreak and determined 53 near full-length genome ('whole genome') sequences. These sequences were combined with 718 whole genome and 1,987 VP1-gene publicly available sequences. In 2018, circulating strains clustered into multiple subgroups in the B3 and A2 subclades, with different phylogenetic origins. Clusters in subclade B3 emerged from strains circulating primarily in the US and Europe in 2016, though some had deeper roots linking to Asian strains, while clusters in A2 traced back to strains detected in East Asia in 2015-2016. In 2018, all sequences from the USA formed a distinct subgroup, containing only three non-US samples. Alongside the varied origins of seasonal strains, we found that diversification of these variants begins up to 18 months prior to the first diagnostic detection during a EV-D68 season. EV-D68 displays strong signs of continuous antigenic evolution and all 2018 A2 strains had novel patterns in the putative neutralizing epitopes in the BC- and DE-loops. The pattern in the BC-loop of the USA B3 subgroup had not been detected on that continent before. Patients with EV-D68 in subclade A2 were significantly older than patients with a B3 subclade virus. In contrast to other subclades, the age distribution of A2 is distinctly bimodal and was found primarily among children and in the elderly. We hypothesize that EV-D68's rapid evolution of surface proteins, extensive diversity, and high rate of geographic mixing could be explained by substantial reinfection of adults. Better understanding of evolution and immunity across diverse viral pathogens, including EV-D68 and SARS-CoV-2, is critical to pandemic preparedness in the future.

Early unrecognised SARS-CoV-2 introductions shaped the first pandemic wave, Sweden, 2020.

BackgroundDespite the unprecedented measures implemented globally in early 2020 to prevent the spread of SARS-CoV-2, Sweden, as many other countries, experienced a severe first wave during the COVID-19 pandemic.AimWe investigated the introduction and spread of SARS-CoV-2 into Sweden.MethodsWe analysed stored respiratory specimens (n = 1,979), sampled 7 February-2 April 2020, by PCR for SARS-CoV-2 and sequenced PCR-positive specimens. Sequences generated from newly detected cases and stored positive specimens February-June 2020 (n = 954) were combined with sequences (Sweden: n = 730; other countries: n = 129,913) retrieved from other sources for Nextstrain clade assignment and phylogenetic analyses.ResultsTwelve previously unrecognised SARS-CoV-2 cases were identified: the earliest was sampled on 3 March, 1 week before recognised community transmission. We showed an early influx of clades 20A and 20B from Italy (201/328, 61% of cases exposed abroad) and clades 19A and 20C from Austria (61/328, 19%). Clade 20C dominated the first wave (20C: 908/1,684, 54%; 20B: 438/1,684, 26%; 20A: 263/1,684, 16%), and 800 of 1,684 (48%) Swedish sequences formed a country-specific 20C cluster defined by a spike mutation (G24368T). At the regional level, the proportion of clade 20C sequences correlated with an earlier weighted mean date of COVID-19 deaths.ConclusionCommunity transmission in Sweden started when mitigation efforts still focused on preventing influx. This created a transmission advantage for clade 20C, likely introduced from ongoing cryptic spread in Austria. Therefore, pandemic preparedness should have a comprehensive approach, including capacity for large-scale diagnostics to allow early detection of travel-related cases and community transmission.

Surveillance of tick-borne encephalitis in emerging risk areas in southern Sweden: a retrospective case finding study.

Tick-borne encephalitis (TBE) is an emerging infection causing CNS infection of various severity. Good knowledge of the incidence in the population and defined risk areas is important in risk communication and vaccination recommendations. The aim of this study was to investigate potential underreporting by retrospectively diagnose TBE among patients with viral CNS infections of unknown etiology in a region with emerging risk areas for TBE, and define variables associated with performed TBE serology at the time of infection. Epidemiological data and microbiological diagnostics of cases with viral CNS infection of unknown etiology treated at departments of infectious diseases and pediatrics in Skåne County during 2000-2012 were investigated. Analyses to evaluate variables associated with performed TBE serology at the time of infection were performed. Retrospective TBE serology was performed on stored blood samples when available. TBE serology was already performed at the time of CNS infection in 193 out of 761 cases. Department, type of clinical manifestation, time period of illness, and whether Borrelia serology had been performed were independent variables associated with having had TBE serology performed or not at the time of illness. Only one of 137 cases, where samples could be retrospectively analyzed for TBE, turned out positive. This study shows a low frequency of TBE sampling among patients with meningoencephalitis in a region with emerging risk for TBE. A higher awareness of TBE as differential diagnosis could contribute to earlier detection of new risk areas and adequate preventive advice to the public.

Clinical phenotypes and outcomes of SARS-CoV-2, influenza, RSV and seven other respiratory viruses: a retrospective study using complete hospital data.

BACKGROUND: An understanding of differences in clinical phenotypes and outcomes COVID-19 compared with other respiratory viral infections is important to optimise the management of patients and plan healthcare. Herein we sought to investigate such differences in patients positive for SARS-CoV-2 compared with influenza, respiratory syncytial virus (RSV) and other respiratory viruses. METHODS: We performed a retrospective cohort study of hospitalised adults and children (≤15 years) who tested positive for SARS-CoV-2, influenza virus A/B, RSV, rhinovirus, enterovirus, parainfluenza viruses, metapneumovirus, seasonal coronaviruses, adenovirus or bocavirus in a respiratory sample at admission between 2011 and 2020. RESULTS: A total of 6321 adult (1721 SARS-CoV-2) and 6379 paediatric (101 SARS-CoV-2) healthcare episodes were included in the study. In adults, SARS-CoV-2 positivity was independently associated with younger age, male sex, overweight/obesity, diabetes and hypertension, tachypnoea as well as better haemodynamic measurements, white cell count, platelet count and creatinine values. Furthermore, SARS-CoV-2 was associated with higher 30-day mortality as compared with influenza (adjusted HR (aHR) 4.43, 95% CI 3.51 to 5.59), RSV (aHR 3.81, 95% CI 2.72 to 5.34) and other respiratory viruses (aHR 3.46, 95% CI 2.61 to 4.60), as well as higher 90-day mortality, ICU admission, ICU mortality and pulmonary embolism in adults. In children, patients with SARS-CoV-2 were older and had lower prevalence of chronic cardiac and respiratory diseases compared with other viruses. CONCLUSIONS: SARS-CoV-2 is associated with more severe outcomes compared with other respiratory viruses, and although associated with specific patient and clinical characteristics at admission, a substantial overlap precludes discrimination based on these characteristics.

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.

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.

Outbreak of enterovirus D68 of the new B3 lineage in Stockholm, Sweden, August to September 2016.

We report an enterovirus D68 (EV-D68) outbreak in Stockholm Sweden in 2016. Between 22 August and 25 September EV-D68 was detected in 74/495 respiratory samples analysed at the Karolinska University Hospital. During the peak week, 30/91 (33%) samples were EV-D68 positive. Viral protein (VP)P4/VP2 sequencing revealed that cases were caused by B3 lineage strains. Forty-four (59%) EV-D68-positive patients were children aged ≤ 5 years. Ten patients had severe respiratory or neurological symptoms and one died.

Rapid increase in multidrug-resistant enteric bacilli blood stream infection after prostate biopsy - A 10-year population-based cohort study.

BACKGROUND: Bloodstream infection following a transrectal prostate biopsy is a well-known and feared complication. Previous studies have shown an increase in multi-resistant bacterial infections as a consequence of higher usage of antibiotics in investigated populations. Our aim was to analyze bacterial resistance patterns in positive blood cultures, after prostate biopsies in Stockholm, Sweden, where the use of antibiotics has been low and decreasing during the last 10 years. METHODS: From the three pathology laboratories in Stockholm, reports of prostate examinations were retrieved (n = 56,076) from 2003 to 2012. By linking men to the National Patient Register all but prostate core biopsies were excluded (n = 12,024). Prostate biopsies in men younger than 30 years of age were excluded (n = 5) leaving 44,047 biopsies for analysis. From laboratory information systems data regarding blood cultures were retrieved. Proportions of blood cultures within 30 days by year were calculated. Crude and adjusted logistic regression models were used to estimate ORs. RESULTS: In total, 44,047 prostate biopsies were performed in 32,916 men over 10 years. On 620 occasions a blood culture was drawn within 30 days of the biopsy; 266 of these were positive. The proportions with positive blood cultures in 2003 and 2012 were 0.38 and 1.14%, respectively. The proportion of multidrug-resistant bacteria increased significantly during the study. In the crude and the adjusted analysis, the year of biopsy and Charlson Comorbidity Index were associated with the risk of having a positive blood culture. CONCLUSION: Multidrug-resistant enteric bacilli are becoming a problem in Sweden, despite low antimicrobial use. Men need to be informed about the increasing risks of infectious complications of transrectal prostate biopsy. One out of 50 men undergoing a prostate biopsy will develop symptoms suggestive of a bloodstream infection after the biopsy and one in 100 men will have a positive blood culture.

Structural basis of broad SARS-CoV-2 cross-neutralization by affinity-matured public antibodies.

Descendants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant now account for almost all SARS-CoV-2 infections. The Omicron variant and its sublineages have spike glycoproteins that are highly diverged from the pandemic founder and first-generation vaccine strain, resulting in significant evasion from monoclonal antibody therapeutics and vaccines. Understanding how commonly elicited antibodies can broaden to cross-neutralize escape variants is crucial. We isolate IGHV3-53, using "public" monoclonal antibodies (mAbs) from an individual 7 months post infection with the ancestral virus and identify antibodies that exhibit potent and broad cross-neutralization, extending to the BA.1, BA.2, and BA.4/BA.5 sublineages of Omicron. Deep mutational scanning reveals these mAbs' high resistance to viral escape. Structural analysis via cryoelectron microscopy of a representative broadly neutralizing antibody, CAB-A17, in complex with the Omicron BA.1 spike highlights the structural underpinnings of this broad neutralization. By reintroducing somatic hypermutations into a germline-reverted CAB-A17, we delineate the role of affinity maturation in the development of cross-neutralization by a public class of antibodies.

Neutralisation sensitivity of the SARS-CoV-2 omicron (B.1.1.529) variant: a cross-sectional study.

BACKGROUND: The SARS-CoV-2 omicron (B.1.1.529) variant, which was first identified in November, 2021, spread rapidly in many countries, with a spike protein highly diverged from previously known variants, and raised concerns that this variant might evade neutralising antibody responses. We therefore aimed to characterise the sensitivity of the omicron variant to neutralisation. METHODS: For this cross-sectional study, we cloned the sequence encoding the omicron spike protein from a diagnostic sample to establish an omicron pseudotyped virus neutralisation assay. We quantified the neutralising antibody ID50 (the reciprocal dilution that produces 50% inhibition) against the omicron spike protein, and the fold-change in ID50 relative to the spike of wild-type SARS-CoV-2 (ie, the pandemic founder variant), for one convalescent reference plasma pool (WHO International Standard for anti-SARS-CoV-2 immunoglobulin [20/136]), three reference serum pools from vaccinated individuals, and two cohorts from Stockholm, Sweden: one comprising previously infected hospital workers (17 sampled in November, 2021, after vaccine rollout and nine in June or July, 2020, before vaccination) and one comprising serum from 40 randomly sampled blood donors donated during week 48 (Nov 29-Dec 5) of 2021. Furthermore, we assessed the neutralisation of omicron by five clinically relevant monoclonal antibodies (mAbs). FINDINGS: Neutralising antibody responses in reference sample pools sampled shortly after infection or vaccination were substantially less potent against the omicron variant than against wild-type SARS-CoV-2 (seven-fold to 42-fold reduction in ID50 titres). Similarly, for sera obtained before vaccination in 2020 from a cohort of convalescent hospital workers, neutralisation of the omicron variant was low to undetectable (all ID50 titres <20). However, in serum samples obtained in 2021 from two cohorts in Stockholm, substantial cross-neutralisation of the omicron variant was observed. Sera from 17 hospital workers after infection and subsequent vaccination had a reduction in average potency of only five-fold relative to wild-type SARS-CoV-2 (geometric mean ID50 titre 495 vs 105), and two donors had no reduction in potency. A similar pattern was observed in randomly sampled blood donors (n=40), who had an eight-fold reduction in average potency against the omicron variant compared with wild-type SARS-CoV-2 (geometric mean ID50 titre 369 vs 45). We found that the omicron variant was resistant to neutralisation (50% inhibitory concentration [IC50] >10 µg/mL) by mAbs casirivimab (REGN-10933), imdevimab (REGN-10987), etesevimab (Ly-CoV016), and bamlanivimab (Ly-CoV555), which form part of antibody combinations used in the clinic to treat COVID-19. However, S309, the parent of sotrovimab, retained most of its activity, with only an approximately two-fold reduction in potency against the omicron variant compared with ancestral D614G SARS-CoV-2 (IC50 0·1-0·2 µg/mL). INTERPRETATION: These data highlight the extensive, but incomplete, evasion of neutralising antibody responses by the omicron variant, and suggest that boosting with licensed vaccines might be sufficient to raise neutralising antibody titres to protective levels. FUNDING: European Union Horizon 2020 research and innovation programme, European and Developing Countries Clinical Trials Partnership, SciLifeLab, and the Erling-Persson Foundation.

Interactions between seasonal human coronaviruses and implications for the SARS-CoV-2 pandemic: A retrospective study in Stockholm, Sweden, 2009-2020.

OBJECTIVES: The four seasonal coronaviruses 229E, NL63, OC43, and HKU1 are frequent causes of respiratory infections and show annual and seasonal variation. Increased understanding about these patterns could be informative about the epidemiology of SARS-CoV-2. METHODS: Results from PCR diagnostics for the seasonal coronaviruses, and other respiratory viruses, were obtained for 55,190 clinical samples analyzed at the Karolinska University Hospital, Stockholm, Sweden, between 14 September 2009 and 2 April 2020. RESULTS: Seasonal coronaviruses were detected in 2130 samples (3.9 %) and constituted 8.1 % of all virus detections. OC43 was most commonly detected (28.4 % of detections), followed by NL63 (24.0 %), HKU1 (17.6 %), and 229E (15.3 %). The overall fraction of positive samples was similar between seasons, but at species level there were distinct biennial alternating peak seasons for the Alphacoronaviruses, 229E and NL63, and the Betacoronaviruses, OC43 and HKU1, respectively. The Betacoronaviruses peaked earlier in the winter season (Dec-Jan) than the Alphacoronaviruses (Feb-Mar). Coronaviruses were detected across all ages, but diagnostics were more frequently requested for paediatric patients than adults and the elderly. OC43 and 229E incidence was relatively constant across age strata, while that of NL63 and HKU1 decreased with age. CONCLUSIONS: Both the Alphacoronaviruses and Betacoronaviruses showed alternating biennial winter incidence peaks, which suggests some type of immune mediated interaction. Symptomatic reinfections in adults and the elderly appear relatively common. Both findings may be of relevance for the epidemiology of SARS-CoV-2.

Potential impact of seasonal forcing on a SARS-CoV-2 pandemic.

A novel coronavirus (SARS-CoV-2) first detected in Wuhan, China, has spread rapidly since December 2019, causing more than 100,000 confirmed infections and 4000 fatalities (as of 10 March 2020). The outbreak has been declared a pandemic by the WHO on Mar 11, 2020. Here, we explore how seasonal variation in transmissibility could modulate a SARS-CoV-2 pandemic. Data from routine diagnostics show a strong and consistent seasonal variation of the four endemic coronaviruses (229E, HKU1, NL63, OC43) and we parameterise our model for SARS-CoV-2 using these data. The model allows for many subpopulations of different size with variable parameters. Simulations of different scenarios show that plausible parameters result in a small peak in early 2020 in temperate regions of the Northern Hemisphere and a larger peak in winter 2020/2021. Variation in transmission and migration rates can result in substantial variation in prevalence between regions. While the uncertainty in parameters is large, the scenarios we explore show that transient reductions in the incidence rate might be due to a combination of seasonal variation and infection control efforts but do not necessarily mean the epidemic is contained. Seasonal forcing on SARS-CoV-2 should thus be taken into account in the further monitoring of the global transmission. The likely aggregated effect of seasonal variation, infection control measures, and transmission rate variation is a prolonged pandemic wave with lower prevalence at any given time, thereby providing a window of opportunity for better preparation of health care systems.

Comparative evaluation of CLIA and EIA for Quantiferon-TB Gold Plus.

The purpose of this study was to compare the Quantiferon-TB Gold Plus test analysed with chemiluminescence immunoassay (CLIA) to immunoassay (EIA). One hundred and twenty-five clinical specimens submitted to Karolinska University Hospital for Quantiferon-TB Gold Plus analysis were used to analyse agreement of the CLIA and EIA assays. The imprecision on CLIA was determined by analysis of pooled clinical samples of antigen tubes (TB1 and TB2), a reference material from National Institute for Biological Standards and Control (NIBSC 82/587) and two controls of the test. Recovery on CLIA was determined by analysis of the TB1 and TB2 samples and NIBSC 82/587. Concordant results were obtained for 110 (88.0%) of 125 samples with cut-off including a borderline range. With no borderline range, 121 of 125 samples (96.8%) showed concordant results. Repeatability had a coefficient of variation (CV) of 14.9%. Reproducibility for pooled clinical samples TB1 and TB2 had a CV of 10.5% and 11.0%, respectively, and for the NIBSC 82/587 7.45%, and kit controls 31.0% respective 12.2%. Overall recovery had a mean of 94.1% (SD 12%). We concluded that Quantiferon-TB Gold Plus analysed with the CLIA assay shows good concordance with EIA and acceptable imprecision.

Intra- and interpatient evolution of enterovirus D68 analyzed by whole-genome deep sequencing.

Worldwide outbreaks of enterovirus D68 (EV-D68) in 2014 and 2016 have caused serious respiratory and neurological disease. To investigate diversity, spread, and evolution of EV-D68 we performed near full-length deep sequencing in fifty-four samples obtained in Sweden during the 2014 and 2016 outbreaks. In most samples, intrapatient variability was low and dominated by rare synonymous variants, but three patients showed evidence of dual infections with distinct EV-D68 variants from the same subclade. Interpatient evolution showed a very strong temporal signal, with an evolutionary rate of 0.0039 ± 0.0001 substitutions per site and year. Phylogenetic trees reconstructed from the sequences suggest that EV-D68 was introduced into Stockholm several times during the 2016 outbreak. Putative neutralization targets in the BC and DE loops of the VP1 protein were slightly more diverse within-host and tended to undergo more frequent substitution than other genomic regions. However, evolution in these loops did not appear to have been driven the emergence of the 2016 B3-subclade directly from the 2014 B1-subclade. Instead, the most recent ancestor of both clades was dated to 2009. The study provides a comprehensive description of the intra- and interpatient evolution of EV-D68, including the first report of intrapatient diversity and dual infections. The new data along with publicly available EV-D68 sequences are included in an interactive phylodynamic analysis on nextstrain.org/enterovirus/d68 to facilitate timely EV-D68 tracking in the future.

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.

Coexistence of two clades of enterovirus D68 in pediatric Swedish patients in the summer and fall of 2014.

In 2014, an outbreak of enterovirus D68 (EV-D68) was observed in North America, with cases of severe respiratory illness and a possible etiological link to cases of acute flaccid paralysis. EV-D68 has also been reported from European countries, but no data from Sweden are available. This study investigated respiratory specimens collected during July-October 2014 from 30 Swedish children aged 0-9 years who were positive for enterovirus and/or rhinovirus in routine clinical PCR. Seven samples were typed as EV-D68 by VP4/VP2 sequencing. Two genetically distinct EV-D68 variants coexisted. Six viruses belonged to clade B, the variant involved in the North American outbreak, and one virus belonged to clade A. Respiratory illness was the major symptom among EV-D68 infected patients and all fully recovered. This is the first report of EV-D68 in Sweden. Considering the current epidemiological situation, genotyping and specific EV-D68 testing should be considered in patients with severe respiratory illness who test positive for enterovirus or rhinovirus in routine diagnostics.