Epidemiological and clinical insights into the enterovirus D68 upsurge in Europe 2021/22 and the 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 and its clinical impact during the fall-winter season of 2021/22. From 19 European countries, 58 institutes reported 10,481 (6.8%) EV-positive samples of which 1,004 (9.6%) were identified as EV-D68 (852 respiratory samples). Clinical data was reported for 969 cases. 78.9% of infections were reported in children (0-5 years); 37.9% of cases were hospitalised. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases with six reported with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of two novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale EV-D68 European upsurge with severe clinical impact and the emergence of B3-derived lineages.

Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

Metagenomic sequencing, molecular characterization, and Bayesian phylogenetics of imported type 2 vaccine-derived poliovirus, Spain, 2021.

Introduction: In 2021, a type 2 vaccine-derived poliovirus (VDPV2) was isolated from the stool of a patient with acute flaccid paralysis (AFP) admitted to Spain from Senegal. A virological investigation was conducted to characterize and trace the origin of VDPV2. Methods: We used an unbiased metagenomic approach for the whole-genome sequencing of VDPV2 from the stool (pre-treated with chloroform) and from the poliovirus-positive supernatant. Phylogenetic analyses and molecular epidemiological analyses relying on the Bayesian Markov Chain Monte Carlo methodology were used to determine the geographical origin and estimate the date of the initiating dose of the oral poliovirus vaccine for the imported VDPV2. Results: We obtained a high percentage of viral reads per total reads mapped to the poliovirus genome (69.5% for pre-treated stool and 75.8% for isolate) with a great depth of sequencing coverage (5,931 and 11,581, respectively) and complete genome coverage (100%). The two key attenuating mutations in the Sabin 2 strain had reverted (A481G in the 5'UTR and Ile143Thr in VP1). In addition, the genome had a recombinant structure between type-2 poliovirus and an unidentified non-polio enterovirus-C (NPEV-C) strain with a crossover point in the protease-2A genomic region. VP1 phylogenetic analysis revealed that this strain is closely related to VDPV2 strains circulating in Senegal in 2021. According to Bayesian phylogenetics, the most recent common ancestor of the imported VDPV2 could date back 2.6 years (95% HPD: 1.7-3.7) in Senegal. We suggest that all VDPV2s circulating in 2020-21 in Senegal, Guinea, Gambia, and Mauritania have an ancestral origin in Senegal estimated around 2015. All 50 stool samples from healthy case contacts collected in Spain (n = 25) and Senegal (n = 25) and four wastewater samples collected in Spain were poliovirus negative. Discussion: By using a whole-genome sequencing protocol with unbiased metagenomics from the clinical sample and viral isolate with high sequence coverage, efficiency, and throughput, we confirmed the classification of VDPV as a circulating type. The close genomic linkage with strains from Senegal was consistent with their classification as imported. Given the scarce number of complete genome sequences for NPEV-C in public databases, this protocol could help expand poliovirus and NPEV-C sequencing capacity worldwide.

Invasive bacterial infection in children with fever and petechial rash in the emergency department: a national prospective observational study.

OBJECTIVE: To determine the incidence and clinical predictors of invasive bacterial infection (IBI) in well-appearing children who present to the emergency department (ED) with fever and petechiae. DESIGN: A prospective, observational, multicentre study was conducted in 18 hospitals between November 2017 and October 2019. PATIENTS: A total of 688 patients were recruited. MAIN OUTCOME MEASURES: The primary outcome was the presence of IBI. Clinical features and laboratory test results were described and related to the presence of IBI. RESULTS: Ten IBIs were found (1.5%), comprising eight cases of meningococcal disease and two of occult pneumococcal bacteraemia. Median age was 26.2 months (IQR 15.3-51.2). Blood samples were obtained from 575 patients (83.3%). Patients with an IBI had a shorter time from fever to ED visit (13.5 hours vs 24 hours) and between fever and rash onset (3.5 hours vs 24 hours). Values for absolute leucocyte count, total neutrophil count, C reactive protein and procalcitonin were significantly higher in patients with an IBI. Significantly fewer patients with a favourable clinical status while in the observation unit were found to have an IBI (2/408 patients, 0.5%) than when clinical status was unfavourable (3/18, 16.7%). CONCLUSIONS: The incidence of IBI among children with fever and petechial rash is lower than previously reported (1.5%). The time from fever to ED visit and to rash onset was shorter in patients with an IBI. Patients with a favourable clinical course during observation in the ED are at lower risk of IBI.

Impact of the bacterial nasopharyngeal microbiota on the severity of genus enterovirus lower respiratory tract infection in children: A case-control study.

INTRODUCTION: Rhinoviruses (RV) and enteroviruses (EV) are among the main causative etiologies of lower respiratory tract infection (LRTI) in children. The clinical spectrum of RV/EV infection is wide, which could be explained by diverse environmental, pathogen-, and host-related factors. Little is known about the nasopharyngeal microbiota as a risk factor or disease modifier for RV/EV infection in pediatric patients. This study describes distinct nasopharyngeal microbiota profiles according to RV/EV LRTI status in children. METHODS: Cross-sectional case-control study, conducted at Hospital Sant de Déu (Barcelona, Spain) from 2017 to 2020. Three groups of children <5 years were included: healthy controls without viral detection (Group A), mild or asymptomatic controls with RV/EV infection (Group B), and cases with severe RV/EV infection admitted to the pediatric intensive care unit (PICU) (Group C). Nasopharyngeal samples were collected from participants for viral DNA/RNA detection by multiplex-polymerase chain reaction and bacterial microbiota characterization by 16S rRNA gene sequencing. RESULTS: A total of 104 subjects were recruited (A = 17, B = 34, C = 53). Children's nasopharyngeal microbiota composition varied according to their RV/EV infection status. Richness and diversity were decreased among children with severe infection. Nasopharyngeal microbiota profiles enriched in genus Dolosigranulum were related to respiratory health, while genus Haemophilus was specifically predominant in children with severe RV/EV LRTI. Children with mild or asymptomatic RV/EV infection showed an intermediate profile. CONCLUSIONS: These results suggest a close relationship between the nasopharyngeal microbiota and different clinical presentations of RV/EV infection.

Rare Recombinant GI.5[P4] Norovirus That Caused a Large Foodborne Outbreak of Gastroenteritis in a Hotel in Spain in 2021.

Noroviruses are among the most important causes of acute gastroenteritis (AGE). In summer 2021, a large outbreak of norovirus infections affecting 163 patients, including 15 norovirus-confirmed food handlers, occurred in a hotel in Murcia in southeast Spain. A rare GI.5[P4] norovirus strain was identified as the cause of the outbreak. The epidemiological investigation determined that norovirus transmission might have been initiated through an infected food handler. The food safety inspection found that some symptomatic food handlers continued working during illness. Molecular investigation with whole-genome and ORF1 sequencing provided enhanced genetic discrimination over ORF2 sequencing alone and enabled differentiation of the GI.5[P4] strains into separate subclusters, suggesting different chains of transmission. These recombinant viruses have been identified circulating globally over the last 5 years, warranting further global surveillance. IMPORTANCE Due to the large genetic diversity of noroviruses, it is important to enhance the discriminatory power of typing techniques to differentiate strains when investigating outbreaks and elucidating transmission chains. This study highlights the importance of (i) using whole-genome sequencing to ensure genetic differentiation of GI noroviruses to track chains of transmission during outbreak investigations and (ii) the adherence of symptomatic food handlers to work exclusion rules and strict hand hygiene practices. To our knowledge, this study provides the first full-length genome sequences of GI.5[P4] strains apart from the prototype strain.

Norovirus GII.3[P12] Outbreak Associated with the Drinking Water Supply in a Rural Area in Galicia, Spain, 2021.

On 30 September 2021, the city council of Muxia, Spain (population of 4,564 inhabitants), reported an unusual increase of patients with acute gastroenteritis (AGE). Because geographically widespread villages belonging to the same water supply were affected, a waterborne outbreak was suspected. Overall, 115 probable cases were ascertained during epidemiological investigations carried out by the local health authority (attack rate, 5.7%); the age range was 0 to 92 years, and 54% were female. The main symptoms were vomiting (78.1%) and diarrhea (67.5%). Primary cases peaked on 29 September and subsided on 1 October, compatible with a point-source outbreak followed by possible secondary cases until 7 October. We conducted an unmatched case-control study using phone surveys. The case-control study included 62 cases and 46 controls. Univariate analysis showed that cases had a higher exposure to tap water through direct consumption (odds ratio [OR] = 86; 95% confidence interval [CI], 18 to 409) or vegetable washing (OR = 27; 95% CI, 7 to 98). Norovirus GII was detected in two terminal points of the water supply system, and 14 cases were laboratory confirmed after detection of GII in stool samples. A unique genotype (GII.3[P12]) was identified in stool samples. On 1 October, a tap water ban was put in place and the water was purged and chlorinated. The rapid increase in the number of cases and its decline after implementing control measures suggested a waterborne point-source outbreak among the residents of Muxia sharing the same water distribution system. IMPORTANCE Noroviruses are likely to be underrecognized in most suspected waterborne outbreaks. Therefore, effective norovirus detection and the early recognition of water as a possible source of infection are important to reduce morbidity as appropriate steps are taken to control the source. In our study, we combined epidemiological, environmental, and microbiological investigations to demonstrate that it was a waterborne outbreak caused by norovirus. Metagenomic sequencing in one norovirus-positive stool sample confirmed norovirus etiology and the absence of other potential pathogens. Detection of fecal indicator bacteria and the fact that the drinking water was not chlorinated suggest a breakdown in chlorination as the cause of the outbreak. This outbreak investigation also demonstrated the importance of timely communication to the public about the risk linked to tap water consumption.

Molecular characterization of enterovirus detected in cerebrospinal fluid and wastewater samples in Monastir, Tunisia, 2014-2017.

BACKGROUND: Enteroviruses (EVs) are considered the main causative agents responsible for aseptic meningitis worldwide. This study was conducted in the Monastir region of Tunisia in order to know the prevalence of EV infections in children with meningitis symptoms. Detected EV types were compared to those identified in wastewater samples. METHODS: Two hundred CSF samples collected from hospitalized patients suspected of having aseptic meningitis for an EV infection between May 2014 and May 2017 and 80 wastewater samples collected in the same time-period were analyzed. EV detection and genotyping were performed using PCR methods followed by sequencing. Phylogenetic analyses in the 3'-VP1 region were also carried-out. RESULTS: EVs were detected in 12% (24/200) CSF and in 35% (28/80) wastewater samples. EV genotyping was reached in 50% (12/24) CSF-positive samples and in 64% (18/28) sewage. Most frequent types detected in CSF were CVB3, E-30 and E-9 (25% each). In wastewater samples, the same EVs were identified, but also other types non-detected in CSF samples, such as E-17,CVA9 and CVB1 from EV species B, and EV-A71 and CVA8 from EV-A, suggesting their likely lower pathogenicity. Phylogenetic analysis showed that within the same type, different strains circulate in Tunisia. For some of the EV types such as E-9, E-11 or CVB3, the same strains were detected in CSF and wastewater samples. CONCLUSIONS: Epidemiological studies are important for the surveillance of the EV infections and to better understand the emergence of certain types and variants.

Epidemiology of Echovirus 30 Infections Detected in a University Hospital in Catalonia, Spain, in 1995-2020.

There is a growing interest in echovirus 30 (E30), an enterovirus responsible for neurological disease and hospitalization. There are multiple studies of outbreaks, but few that study the epidemiology over long periods of time. Our study aims to describe the clinical, epidemiological and microbiological characteristics of a series of E30 infections detected over 26 years. Data were retrospectively collected from a database of all enterovirus infections identified in our laboratory. They were detected by viral isolation or nucleic acid detection in patients presenting with respiratory or neurological infections, rash, sepsis-like syndrome, or gastroenteritis. Enterovirus genotyping was performed by amplification of the VP1 gene using RT-nested PCR, followed by sequencing and BLAST analysis. Of the 2402 enterovirus infections detected, 1619 were linked to at least one genotype and 173 were caused by E30. Clinical information was available for 158 (91.3%) patients. E30 was associated with neurological infection in 107 (67.8%) cases and it was detected almost every year. Phylogenetic analysis was performed with 67 sequences. We observed that E30 strains circulating in Catalonia from 1996 to 2016 belong to two lineages (E and F), although the majority cluster was in F. In 2018, lineage I emerged as the dominant lineage.

An imported case of vaccine-derived poliovirus type 2, Spain in the context of the ongoing polio Public Health Emergency of International Concern, September 2021.

The monthly retrospective search for unreported acute flaccid paralysis (AFP) cases conducted as a complementary component of the Spanish AFP surveillance system identified a case of AFP in a child admitted in Spain from Senegal during August 2021. Vaccine-derived poliovirus 2 was identified in the stool in September 2021. We present public health implications and response undertaken within the framework of the National Action Plan for Polio Eradication and the Public Health Emergency of International Concern.

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.

Genomic surveillance of enterovirus associated with aseptic meningitis cases in southern Spain, 2015-2018.

New circulating Enterovirus (EV) strains often emerge through recombination. Upsurges of recombinant non-polio enteroviruses (NPEVs) associated with neurologic manifestations such as EVA71 or Echovirus 30 (E30) are a growing public health concern in Europe. Only a few complete genomes of EVs circulating in Spain are available in public databases, making it difficult to address the emergence of recombinant EVs, understand their evolutionary relatedness and the possible implication in human disease. We have used metagenomic (untargeted) NGS to generate full-length EV genomes from CSF samples of EV-positive aseptic meningitis cases in Southern Spain between 2015 and 2018. Our analyses reveal the co-circulation of multiple Enterovirus B (EV-B) types (E6, E11, E13 and E30), including a novel E13 recombinant form. We observed a genetic turnover where emergent lineages (C1 for E6 and I [tentatively proposed in this study] for E30) replaced previous lineages circulating in Spain, some concomitant with outbreaks in other parts of Europe. Metagenomic sequencing provides an effective approach for the analysis of EV genomes directly from PCR-positive CSF samples. The detection of a novel, disease-associated, recombinant form emphasizes the importance of genomic surveillance to monitor spread and evolution of EVs.

Lower Respiratory Tract Infection and Genus Enterovirus in Children Requiring Intensive Care: Clinical Manifestations and Impact of Viral Co-Infections.

Infection by rhinovirus (RV) and enterovirus (EV) in children ranges from asymptomatic infection to severe lower respiratory tract infection (LRTI). This cohort study evaluates the clinical impact of RV/EV species, alone or in codetection with other viruses, in young children with severe LRTI. Seventy-one patients aged less than 5 years and admitted to the Paediatric Intensive Care Unit (PICU) of a reference children's hospital with RV or EV (RV/EV) LRTI were prospectively included from 1/2018 to 3/2020. A commercial PCR assay for multiple respiratory pathogens was performed in respiratory specimens. In 22/71, RV/EV + respiratory syncytial virus (RSV) was found, and 18/71 had RV/EV + multiple viral detections. Patients with single RV/EV detection required invasive mechanical ventilation (IMV) as frequently as those with RSV codetection, whereas none of those with multiple viral codetections required IMV. Species were determined in 60 samples, 58 being RV. No EV-A, EV-C, or EV-D68 were detected. RV-B and EV-B were only found in patients with other respiratory virus codetections. There were not any associations between RV/EV species and severity outcomes. To conclude, RV/EV detection alone was observed in young children with severe disease, while multiple viral codetections may result in reduced clinical severity. Differences in pathogenicity between RV and EV species could not be drawn.

Neurodevelopmental Outcomes of Infants Younger Than 90 Days Old Following Enterovirus and Parechovirus Infections of the Central Nervous System.

Enteroviruses (EVs) and human parechoviruses (HPeVs) are a major cause of central nervous system (CNS) infection in young infants. They have been implicated in neurodevelopmental delay, however limited data are available. The aim of this study is to describe the clinical outcome of young infants and to assess and compare the medium-term neurodevelopment following CNS infections caused by EV and HPeV. A multicentre observational ambispective study was conducted between May 2013 and March 2018. Children under 3 months of age with EV or HPeV CNS infection excluding encephalitis were included. Infants were contacted 1 year after the acute infection and their neurological development was evaluated using the Ages and Stages Questionnaire-3 (ASQ-3). If any area assessed was abnormal during the first round of tests, a second round was completed 6 to 12 months later. Forty-eight young infants with EV and HPeV CNS infection were identified: 33 (68.8%) were positive for EV and 15 (31.3%) for HPeV. At first assessment 14 out of 29 EV (48.3%) and 3 out of 15 HPeV (20%) positive cases presented some developmental concern in the ASQ-3 test. EV-positive infants showed mild and moderate alteration in all domains analyzed and HPeV-positive infants showed mild alterations only in gross and fine motor domains. Significant alterations in communication were observed in EV-positive but not in HPeV-positive infants (31 vs. 0%, p = 0.016). At second assessment 4 out of 13 EV-positive patients (30.8%) showed mild to moderate concerns in communication and gross motor function domains and 3 out of 13 (23.1%) showed significant concern in fine motor function. Although CNS infections without associated encephalitis are generally assumed to be benign our study shows that at a median age of 18 months almost half of the EV-infected infants (48.3%) and 20% of HPeV-positive infants presented some developmental concern in the ASQ-3 test. We recommend monitor the neurological development of infants during the first years of life after HPeV CNS infection and especially after EV CNS infection, even in mild cases, for an early intervention and stimulation of psychomotor development if necessary.

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.