[Importance of enteroviruses in neuropaediatrics: from polioviruses to other enteroviruses]. / Importancia de los enterovirus en neuropediatria: de los poliovirus a otros enterovirus.

Enteroviruses are among the most common human viruses around the world. More than 100 different serotypes that can cause a range of clinical pathologies have been identified, although the most frequent are those that affect the central nervous system, such as aseptic meningitis, encephalitis or paralysis, which in some cases can be very severe or even fatal. In recent years, enterovirus outbreaks associated to new diseases have been reported all over the world, and as a result some serotypes have been considered 'emerging' pathogens. Yet, our knowledge about these viruses, especially about the non-polio enteroviruses that produce neuropathologies, is still limited. Surveillance systems are crucial to understand the epidemiology of the infections due to enterovirus and to be able to take appropriate action to deal with future outbreaks or epidemics.

TITLE: Importancia de los enterovirus en neuropediatria: de los poliovirus a otros enterovirus.Los enterovirus son unos de los virus humanos mas comunes en todo el mundo. Se han identificado mas de 100 serotipos distintos que pueden causar diversas patologias clinicas, aunque las mas frecuentes son las que afectan al sistema nervioso central, como meningitis aseptica, encefalitis o paralisis, que en algunos casos pueden ser muy graves e incluso mortales. En los ultimos años, se han descrito brotes por enterovirus asociados a nuevas enfermedades en todo el mundo, de tal manera que algunos serotipos se consideran patogenos 'emergentes'. Sin embargo, el conocimiento que tenemos sobre estos virus, especialmente sobre los enterovirus no polio que producen neuropatologias, todavia es limitado. Los sistemas de vigilancia resultan fundamentales para entender la epidemiologia de las infecciones por enterovirus y poder prevenir y actuar frente a futuros brotes o epidemias.

Transporte pediátrico y neonatal en España, Portugal y Latinoamérica / Pediatric and neonatal transport in Spain, Portugal and Latin America

OBJETIVO: Estudiar la organización del transporte interhospitalario pediátrico y neonatal en la península ibérica y Latinoamérica. DISEÑO: Estudio observacional prospectivo mediante una encuesta enviada por correo electrónico con preguntas sobre el sistema de transporte nacional, regional y local, el tipo y vehículo de traslado, el material y el personal y su formación. Ámbito: Responsables pediátricos de hospitales de España, Portugal y Latinoamérica. RESULTADOS: Se analizaron 117 encuestas provenientes de 15 países. De ellas, 55 (47%) procedían de 15 comunidades autónomas de España y el resto de Portugal y de 13 países latinoamericanos. Muy pocas regiones y ciudades tienen un sistema de transporte pediátrico y neonatal específico. El transporte solo está unificado en las comunidades españolas de Baleares y Cataluña y en Portugal. En Chile el sistema de transporte es mixto, pediátrico y del adulto. Solo un 51,4% de los hospitales tiene un sistema de formación del personal de transporte, y solo en el 36,4% la formación es específica en transporte pediátrico. En España y Portugal los sistemas de transporte son fundamentalmente públicos, mientras que en Latinoamérica coexisten sistemas públicos y privados. Los equipos de transporte de la península ibérica tienen más material pediátrico y neonatal y reciben más formación en transporte pediátrico que los de Latinoamérica. CONCLUSIONES: Existe una gran variabilidad en la organización del transporte pediátrico en cada país y región. En la mayoría de los países y ciudades no existe un sistema unificado y específico de transporte pediátrico con un personal cualificado y un material de traslado específico

OBJECTIVE: To study the organization of inter-hospital transport of pediatric and neonatal patients in Spain, Portugal and Latin America. DESIGN: An observational study was performed. An on-line survey was sent by email including questions about characteristics of national, regional and local health transport systems, vehicles, material, and composition of the transport team and their training. SETTING: Hospital pediatric healthcare professionals treating children in Spain, Portugal and Latin America RESULTS: A total of 117 surveys from 15 countries were analyzed. Of them, 55 (47%) come from 15 regions of Spain and the rest from Portugal and 13 Latin American countries. The inter-hospital transport of pediatric patients is unified only in the Spanish regions of Baleares and Cataluña and in Portugal. Chile has a mixed unified transport system for pediatric and adult patients. Only 51.4% of responders have an educational program for the transport personnel, and only in 36.4% of them the educational program is specific for pediatric patients. In Spain and Portugal the transport is executed mostly by public entities, while in Latin America public and private systems coexist. Specific pediatric equipment is more frequent in the transport teams in the Iberian Peninsula than in Latin American teams. The specific pediatric transport training is less frequent for teams in Latin America than on Spain and Portugal. CONCLUSIONS: There is a great variation in the organization of children transport in each country and region. Most of countries and cities do not have unified and specific teams of pediatric transport, with pediatric qualified personnel and specific material

Outbreak of brainstem encephalitis associated with enterovirus-A71 in Catalonia, Spain (2016): a clinical observational study in a children's reference centre in Catalonia.

OBJECTIVES: To describe the characteristics of an outbreak of brainstem encephalitis and encephalomyelitis related to enterovirus (EV) infection in Catalonia (Spain), a setting in which these manifestations were uncommon. METHODS: Clinical and microbiological data were analysed from patients with neurological symptoms associated with EV detection admitted to a reference paediatric hospital between April and June 2016. RESULTS: Fifty-seven patients were included. Median age was 27.7 months (p25-p75 17.1-37.6). Forty-one (72%) were diagnosed with brainstem encephalitis, seven (12%) with aseptic meningitis, six (11%) with encephalitis, and three (5%) with encephalomyelitis (two out of three with cardiopulmonary failure). Fever, lethargy, and myoclonic jerks were the most common symptoms. Age younger than 12 months, higher white-blood-cell count, and higher procalcitonin levels were associated with cardiopulmonary failure. Using a PAN-EV real-time PCR, EV was detected in faeces and/or nasopharyngeal aspirate in all the patients, but it was found in cerebrospinal fluid only in patients with aseptic meningitis. EV was genotyped in 47 out of 57 and EV-A71 was identified in 40 out of 47, being the only EV type found in patients with brainstem symptoms. Most of the detected EV-A71 strains were subgenogroup C1. Intravenous immunoglobulins were used in 34 patients. Eight cases (14%) were admitted to the intensive care unit. All the patients but three, those with encephalomyelitis, showed a good clinical course and had no significant sequelae. No deaths occurred. CONCLUSIONS: The 2016 outbreak of brainstem encephalitis in Catalonia was associated with EV-A71 subgenogroup C1. Despite the clinical manifestations of serious disease, a favourable outcome was observed in the majority of patients.

Pediatric and neonatal transport in Spain, Portugal and Latin America. / Transporte pediátrico y neonatal en España, Portugal y Latinoamérica.

OBJECTIVE: To study the organization of inter-hospital transport of pediatric and neonatal patients in Spain, Portugal and Latin America. DESIGN: An observational study was performed. An on-line survey was sent by email including questions about characteristics of national, regional and local health transport systems, vehicles, material, and composition of the transport team and their training. SETTING: Hospital pediatric healthcare professionals treating children in Spain, Portugal and Latin America RESULTS: A total of 117 surveys from 15 countries were analyzed. Of them, 55 (47%) come from 15 regions of Spain and the rest from Portugal and 13 Latin American countries. The inter-hospital transport of pediatric patients is unified only in the Spanish regions of Baleares and Cataluña and in Portugal. Chile has a mixed unified transport system for pediatric and adult patients. Only 51.4% of responders have an educational program for the transport personnel, and only in 36.4% of them the educational program is specific for pediatric patients. In Spain and Portugal the transport is executed mostly by public entities, while in Latin America public and private systems coexist. Specific pediatric equipment is more frequent in the transport teams in the Iberian Peninsula than in Latin American teams. The specific pediatric transport training is less frequent for teams in Latin America than on Spain and Portugal. CONCLUSIONS: There is a great variation in the organization of children transport in each country and region. Most of countries and cities do not have unified and specific teams of pediatric transport, with pediatric qualified personnel and specific material.

Optimización de la metodología de cultivo de condrocitos de discos procedentes de hernias discales y comprobación de la presencia del Toll-like receptor 4 (TLR 4) / Optimization of methods of chondrocyte cell culture from human disc hernia subjects and testing the presence of Toll-like receptor 4 (TLR 4)

Objetivo: Optimizar la técnica de cultivo de células del disco intervertebral en humanos y confirmar la expresión del Toll-like receptor 4 (TLR4 ). Material y método: Se cultivaron muestras de núcleo pulposo obtenidas durante la cirugía de hernia discal. Las células cultivadas de los nueve pacientes (todos con degeneración del disco moderada-grave en la escala de Pfirrmann) fueron observadas para determinar su capacidad de crecimiento. Las células cultivadas obtenidas se estudiaron con el fin de determinar su fenotipo mediante inmunotinción y rtPCR. La presencia de TLR4 fue comprobada por los mismos métodos. Resultados: Las células aisladas se sembraron a diferentes concentraciones. La concentración ideal se obtuvo para las condiciones óptimas del cultivo. Se confirmó que el fenotipo de las células fue condrocitario y se confirmó la presencia del receptor TLR4 en las células cultivadas. Conclusión: Se confirma la presencia tanto de ARNm como de la proteína del receptor TLR4 en los condrocitos del disco intervertebral. Este hallazgo allana el camino para la caracterización de las funciones de este receptor en los procesos inflamatorios de la hernia de disco (AU)

Objective: To optimize the technique of culturing human intervertebral disc cells, and to confirm the expression of toll-like receptor 4 (TLR4) in these cells. Material and method: Samples of nucleus pulposus obtained during disc hernia surgery were cultured. Cells from nine patients (all with moderate-severe disc degeneration scores, based on the Pfirmann scale) were followed up to determine their growing capacity. The obtained cultured cells were tested for the chondrocyte phenotype by immunostaining and rt-PCR and the presence of TLR4 was tested by the same methods. Results: The cells were isolated and seeded at different concentrations. The ideal concentration was obtained for optimal culture conditions. The cells were confirmed to have the chondrocyte phenotype and TLR4 was confirmed to be present in the cultured cells. Conclusion: This study confirms the presence of both mRNA and TLR4 protein in intervertebral disc chondrocytes. This paves the way for elucidating of the roles of this receptor in the inflammatory processes of disc hernia (AU)

Infección por parechovirus 3 en un neonato con fiebre y sospecha de sepsis / Human parechovirus-3 infection in a neonate with fever and suspected sepsis

Los parechovirus humanos (HPeV) son virus de la familia Picornaviridae recientemente descritos y causantes de numerosas infecciones en niños pequeños. La afección asociada a estos virus se está empezando a conocer. El HPeV tipo 3 se ha descrito especialmente asociado a síndromes febriles, sepsis-like, meningitis y encefalitis en lactantes muy pequeños y neonatos. Presentamos el caso de una niña de 14 días de vida con un cuadro de fiebre que precisó hospitalización por sospecha de sepsis y en la que se identificó en líquido cefalorraquídeo un HPeV-3. Las pruebas analíticas (leucocitos, fórmula diferencial y procalcitonina en sangre) fueron normales. Los cultivos bacterianos de sangre, orina y líquido cefalorraquídeo fueron estériles. La paciente evolucionó favorablemente. Este caso ilustra la utilidad de investigar la infección por parechovirus en los recién nacidos con fiebre o sospecha de sepsis

The human parechovirus (HPeV) are viruses of the recently described Picornaviridae family and are causing several infections in young children. The pathology associated with these viruses is beginning to emerge. The HPeV type 3, has been described particularly in association with sepsis-like febrile syndromes, meningitis and encephalitis in very young infants and neonates. We report the case of a 14-day-old girl with a fever and clinical sepsis that required hospitalization and in which HPeV-3 was identified in the cerebrospinal fluid. The blood, urine and cerebrospinal fluid bacterial cultures were negative, and the patient improved. This case illustrates the usefulness of investigating parechovirus infection in neonates with fever or suspected sepsis

Eczema coxsackium (Coxsackievirus A6) en una paciente adulta infectada por el virus de la inmunodeficiencia humana / Eczema coxsackium (Coxsackievirus A6) in an human immunodeficiency virus infected adult patient

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[Human parechovirus-3 infection in a neonate with fever and suspected sepsis]. / Infección por parechovirus 3 en un neonato con fiebre y sospecha de sepsis.

The human parechovirus (HPeV) are viruses of the recently described Picornaviridae family and are causing several infections in young children. The pathology associated with these viruses is beginning to emerge. The HPeV type 3, has been described particularly in association with sepsis-like febrile syndromes, meningitis and encephalitis in very young infants and neonates. We report the case of a 14-day-old girl with a fever and clinical sepsis that required hospitalization and in which HPeV-3 was identified in the cerebrospinal fluid. The blood, urine and cerebrospinal fluid bacterial cultures were negative, and the patient improved. This case illustrates the usefulness of investigating parechovirus infection in neonates with fever or suspected sepsis.

Comparative reliability analysis of publicly available software packages for automatic intracranial volume estimation.

Intracranial volume is an important measure in brain research often used as a correction factor in inter subject studies. The current study investigates the resulting outcome in terms of the type of software used for automatically estimating ICV measure. Five groups of 70 subjects are considered, including adult controls (AC) (n=11), adult with dementia (AD) (n=11), pediatric controls (PC) (n=18) and two groups of pediatric epilepsy subjects (PE1.5 and PE3) (n=30) using 1.5 T and 3T scanners, respectively. Reference measurements were calculated for each subject by manually tracing intracranial cavity without sub-sampling. Four publicly available software packages (AFNI, Freesurfer, FSL, and SPM) were examined in their ability to automatically estimate ICV across the five groups. Linear regression analyses suggest that reference measurement discrepancy could be explained best by SPM [R(2)= 0.67;p <; 0.01] for the AC group, Freesurfer [R(2) = 0.46; p = 0.02] for the AD group, AFNI [R(2)=0.97;p<; 0.01] for the PC group and FSL [R(2) = 0.6; p = 0.1] for the PE1.5 and [R(2) = 0.6; p <; 0.01] for PE3 groups. The study demonstrates that the choice of the automated software for ICV estimation is dependent on the population under consideration and whether the software used is atlas-based or not.

Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain.

Hand, foot and mouth disease (HFMD) is a childhood illness frequently caused by genotypes belonging to the enterovirus A species, including coxsackievirus (CV)-A16 and enterovirus (EV)-71. Between 2010 and 2012, several outbreaks and sporadic cases of HFMD occurred in different regions of Spain. The objective of the present study was to describe the enterovirus epidemiology associated with HFMD in the country. A total of 80 patients with HFMD or atypical rash were included. Detection and typing of the enteroviruses were performed directly in clinical samples using molecular methods. Enteroviruses were detected in 53 of the patients (66%). CV-A6 was the most frequent genotype, followed by CV-A16 and EV-71, but other minority types were also identified. Interestingly, during almost all of 2010, CV-A16 was the only causative agent of HFMD but by the end of the year and during 2011, CV-A6 became predominant, while CV-A16 was not detected. In 2012, however, both CV-A6 and CV-A16 circulated. EV-71 was associated with HFMD symptoms only in three cases during 2012. All Spanish CV-A6 sequences segregated into one major genetic cluster together with other European and Asian strains isolated between 2008 and 2011, most forming a particular clade. Spanish EV-71 strains belonged to subgenogroup C2, as did most of the European sequences circulated. In conclusion, the recent increase of HFMD cases in Spain and other European countries has been due to a larger incidence of circulating species A enteroviruses, mainly CV-A6 and CV-A16, and the emergence of new genetic variants of these viruses.

Molecular characterization of enteroviruses associated with neurological infections in Spain, 2008.

In order to investigate the etiology of viral neurological infections in Spain, a national study was performed in 2008. The results obtained have been published. Enteroviruses were the most frequent cause of the aseptic meningitis and infant febrile syndromes. The present report supplements the previous study with the genotyping of the detected enteroviruses. Typing was by amplification of partial VP1 region and sequencing in 70 (53%) of the 132 available cerebrospinal fluid samples positive for enteroviruses. Twelve different genotypes within the B species were identified. Echovirus 4 was predominant (24%), followed by echovirus 30 (19%), echovirus 9 (17%), and echovirus 6 (14%). In summary, a co-circulation of several enterovirus types associated with meningitis in children under 15 years old was observed. Although infrequently detected, echovirus 4 was the predominant genotype identified due to an aseptic meningitis outbreak which occurred in the Canary Islands in 2008.

Viral infections of the central nervous system in Spain: a prospective study.

The aim of the study was to determine the incidence of viruses causing aseptic meningitis, meningoencephalitis, and encephalitis in Spain. This was a prospective study, in collaboration with 17 Spanish hospitals, including 581 cases (CSF from all and sera from 280): meningitis (340), meningoencephalitis (91), encephalitis (76), febrile syndrome (7), other neurological disorders (32), and 35 cases without clinical information. CSF were assayed by PCR for enterovirus (EV), herpesvirus (herpes simplex [HSV], varicella-zoster [VZV], cytomegalovirus [CMV], Epstein-Barr [EBV], and human herpes virus-6 [HHV-6]), mumps (MV), Toscana virus (TOSV), adenovirus (HAdV), lymphocytic choriomeningitis virus (LCMV), West Nile virus (WNV), and rabies. Serology was undertaken when methodology was available. Amongst meningitis cases, 57.1% were characterized; EV was the most frequent (76.8%), followed by VZV (10.3%) and HSV (3.1%; HSV-1: 1.6%; HSV-2: 1.0%, HSV non-typed: 0.5%). Cases due to CMV, EBV, HHV-6, MV, TOSV, HAdV, and LCMV were also detected. For meningoencephalitis, 40.7% of cases were diagnosed, HSV-1 (43.2%) and VZV (27.0%) being the most frequent agents, while cases associated with HSV-2, EV, CMV, MV, and LCMV were also detected. For encephalitis, 27.6% of cases were caused by HSV-1 (71.4%), VZV (19.1%), or EV (9.5%). Other positive neurological syndromes included cerebellitis (EV and HAdV), seizures (HSV), demyelinating disease (HSV-1 and HHV-6), myelopathy (VZV), and polyradiculoneuritis (HSV). No rabies or WNV cases were identified. EVs are the most frequent cause of meningitis, as is HSV for meningoencephalitis and encephalitis. A significant number of cases (42.9% meningitis, 59.3% meningoencephalitis, 72.4% encephalitis) still have no etiological diagnosis.

The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71.

Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.

Onychomadesis after a hand, foot, and mouth disease outbreak in Spain, 2009.

Few reports exist regarding the association between onychomadesis and an enterovirus infection presenting clinically as hand, foot, and mouth disease (HFMD). In February 2009, an outbreak of HFMD occurred in a Spanish nursery school, followed by onychomadesis 36-69 days later. Twelve of 17 children with HFMD developed nail shedding; enterovirus was detected in stool samples from eight (47%) of the 17. However, in only three of the children could an enterovirus serotype coxsackievirus B1 be identified. The epidemiological results of this study confirm onychomadesis as a complication in HFMD. In future outbreaks, molecular characterization of enterovirus from appropriate clinical samples should be studied.

Evolutionary dynamics and temporal/geographical correlates of recombination in the human enterovirus echovirus types 9, 11, and 30.

The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.

Enteroviruses in Spain over the decade 1998-2007: virological and epidemiological studies.

BACKGROUND: Human enteroviruses (HEV) are the commonest cause of viral meningitis as well as other pathologies, therefore HEV characterization is important both in patient management and epidemiological investigation. OBJECTIVES: A 10-year study of patients with enteroviral infection was carried out in Spain to determine the underlying etiology. STUDY DESIGN: HEV were fully typed by microneutralisation tests and/or molecular methods. RESULTS: A collection of 86404 clinical samples were studied in several Spanish laboratories. These were collected from patients with different syndromes, mainly aseptic meningitis (AM), fever, respiratory diseases and acute flaccid paralysis. Of these, 6867 HEV were obtained. At the National Poliovirus Laboratory 2814 were serotypically characterised. Among non-polio enteroviruses, the eight main serotypes were Echovirus 30 (25%), Echovirus 6 (12.4%), Echovirus 13 (8.3%), Echovirus 11 (7.4%) and Echovirus 9 (4.7%), followed by Coxsackievirus B5 (4.2%) and Echovirus 7 and Coxsackievirus A9 (3.7%) each. In AM cases, Echovirus 30 was identified in 39% of them, followed by Echovirus 6 (14%). However, Echovirus 6 was mainly associated with respiratory disease (17%), followed by Echovirus 11 (10%). On the other hand, Echovirus 30, Echovirus 11 and Echovirus 6 contributed equally with 12% of each serotype in the cases of fever. CONCLUSIONS: The present report complements previous data (Trallero et al.(13)), with the results of HEV incidence in Spain from 1998 to 2007. The surveillance described in this study provided valuable information as to which serotypes are in circulation, the emergence of new HEV and association with clinical manifestations.