Surveillance for Enteroviruses Associated with Hand, Foot, and Mouth Disease, and Other Mucocutaneous Symptoms in Spain, 2006-2020.

Hand, foot, and mouth disease (HFMD) is a mild illness caused by enteroviruses (EV), although in some Asian countries, large outbreaks have been reported in the last 25 years, with a considerable incidence of neurological complications. This study describes epidemiological and clinical characteristics of EV infections involved in HFMD and other mucocutaneous symptoms from 2006 to 2020 in Spain. EV-positive samples from 368 patients were included. EV species A were identified in 85.1% of those typed EV. Coxsackievirus (CV) A6 was the prevalent serotype (60.9%), followed by EV-A71 (9.9%) and CVA16 (7.7%). Infections affected children (1-6 years old) mainly, and show seasonality with peaks in spring-summer and autumn. Clinical data indicated few cases of atypical HFMD as well as those with neurological complications (associated with the 2016 EV-A71 outbreak). Phylogenetic analysis of CVA6 VP1 sequences showed different sub-clusters circulating from 2010 to present. In conclusion, HFMD or exanthemas case reporting has increased in Spain in recent years, probably associated with an increase in circulation of CVA6, although they did not seem to show greater severity. However, EV surveillance in mucocutaneous manifestations should be improved to identify the emergence of new types or variants causing outbreaks and more severe pathologies.

Enterovirus D68-associated respiratory and neurological illness in Spain, 2014-2018.

During 2014, enterovirus D68 (EV-D68) outbreaks were described globally, causing severe respiratory diseases in children and, in some cases, subsequent paralysis. In this study, the type characterization of enterovirus (EV) detected in respiratory illnesses and the epidemiology and clinical association of EV-D68 infections in Spain over a five-year period were described. A total of 546 EV-positive samples from hospitalized patients with respiratory infections were included. EV-D68 was the most frequently detected type (46.6%, 191/410 typed EV). Other EV from species A (25.1%), B (27.8%) and C (0.5%) were also identified. EV-D68 infections were more associated with bronchitis while EV-A/B types were more frequent in upper respiratory illness (p < 0.01). EV-D68 was also detected in patients with neurological symptoms (nine meningitis/meningoencephalitis and eight acute flaccid paralysis cases). Phylogenetic analysis of 3'-VP1 region showed most Spanish EV-D68 sequences from 2014 to 2016 belonged to subclades B2/B3, as other American and European strains circulating during the same period. However, those detected in 2017 and 2018 clustered to the emerged subclade D1. In summary, different EV can cause respiratory infections but EV-D68 was the most prevalent, with several strains circulating in Spain at least since 2014. Association between EV-D68 infection and neurological disease was also described.

Molecular epidemiology of an enterovirus A71 outbreak associated with severe neurological disease, Spain, 2016.

IntroductionEnterovirus A71 (EV-A71) is an emerging pathogen that causes a wide range of disorders including severe neurological manifestations. In the past 20 years, this virus has been associated with large outbreaks of hand, foot and mouth disease with neurological complications in the Asia-Pacific region, while in Europe mainly sporadic cases have been reported. In spring 2016, however, an EV-A71 outbreak associated with severe neurological cases was reported in Catalonia and spread further to other Spanish regions.AimOur objective was to investigate the epidemiology and clinical characteristics of the outbreak.MethodsWe carried out a retrospective study which included 233 EV-A71-positive samples collected during 2016 from hospitalised patients. We analysed the clinical manifestations associated with EV-A71 infections and performed phylogenetic analyses of the 3'-VP1 and 3Dpol regions from all Spanish strains and a set of EV-A71 from other countries.ResultsMost EV-A71 infections were reported in children (mean age: 2.6 years) and the highest incidence was between May and July 2016 (83%). Most isolates (218/233) were classified as subgenogroup C1 and 217 of them were grouped in one cluster phylogenetically related to a new recombinant variant strain associated with severe neurological diseases in Germany and France in 2015 and 2016. Moreover, we found a clear association of EV-A71-C1 infection with severe neurological disorders, brainstem encephalitis being the most commonly reported.ConclusionAn emerging recombinant variant of EV-A71-C1 was responsible for the large outbreak in 2016 in Spain that was associated with many severe neurological cases.

Características epidemiológicas y clínicas de los lactantes hospitalizados por infecciones por parechovirus humanos. Estudio prospectivo en España / Epidemiological and clinical characteristics of infants admitted to hospital due to human parechovirus infections: A prospective study in Spain

Introducción: Los parechovirus humanos (HPeV) son virus de la familia Picornaviridae, recientemente descritos, a los que se atribuyen cuadros de fiebre sin foco (FSF), sepsis clínica, gastroenteritis, meningitis o encefalitis fundamentalmente en lactantes pequeños. Nuestro objetivo fue describir la epidemiología y las características clínicas de las infecciones por HPeV en nuestro medio. Pacientes y métodos: Estudio multicéntrico prospectivo, llevado a cabo en 12 hospitales a nivel nacional, entre 2013-2015, en niños < 3 años con FSF, sepsis clínica o patología neurológica. Se realizó determinación de HPeV mediante RT-PCR en el Centro Nacional de Microbiología en suero, heces o líquido cefalorraquídeo. Resultados: Se analizan 47 infecciones por HPeV de un total de 850 muestras (5,52%), siendo HPeV-3 el más frecuente (29 casos), con predominio en mayo y julio, con una distribución bienal. El 57% eran neonatos y solo 2 > 3 meses. Todos los pacientes presentaron fiebre, el 45% irritabilidad, el 18,6% exantema y el 14% diarrea. No se observa ninguna alteración específica en las pruebas bioquímicas. El diagnóstico final más frecuente fue FSF (61%) seguido de sepsis clínica (29%). Aunque un 29% de los niños precisaron ingreso en cuidados intensivos, solo un paciente presentó secuelas. Conclusiones: Los HPeV circulan en nuestro país, afectando fundamentalmente a lactantes < 2 meses y se asocian a FSF y sepsis clínica, con un predominio en primavera y verano. Sería de interés implementar las técnicas moleculares de diagnóstico en todos los hospitales para reconocer y manejar adecuadamente estas infecciones (AU)

Introduction: Human parechovirus (HPeV) is one of the recently described picornaviridae viruses that have been associated with fever of unknown origin (FUO), clinical sepsis, gastroenteritis, meningitis, or encephalitis in very young infants. The aim of this study is to describe the epidemiology and clinical features of these viruses. Patients and methods: A prospective multicentre 3-year study was conducted in 12 hospitals in Spain. Out of 850 specimens examined, 47 were positive (5.52%), with HPeV-3 being the most frequent (29 cases). Infections occurred throughout the year, but mainly in May and July, and a biennial distribution was observed. More than half (57%) were neonates, and only 2 children were older than 3 months. Fever was present in all children, with irritability in 45%, rash in 18.6%, and diarrhoea in 14%. The results of biochemical tests were all in normal range. The most common final diagnosis was FUO (61%), followed by clinical sepsis (29%). Up to 29% of infants were admitted to the intensive care unit, but only one patient had sequelae. Results: Out of 850 specimens examined, 47 were positive (5.52%) for HPeV, with HPeV-3 being the most frequent (29 cases). Infections occurred throughout the year, but mainly in May and July, and a biennial distribution was observed. More than half (57%) were neonates, and only 2 children were older than 3 months. Fever was present in all children, with irritability in 45%, rash in 18.6%, and diarrhoea in 14%. The results of biochemical tests were all in normal range. The most common final diagnosis was FUO (61%), followed by clinical sepsis (29%). Up to 29% of infants were admitted to the intensive care unit, but only one patient had sequelae. Conclusions: HPeV circulates in our country, mainly during spring and summer, and affects young infants with a FUO and clinical sepsis. Molecular diagnostic techniques in all hospitals could help in improving the management of patients with these infections (AU)

[Epidemiological and clinical characteristics of infants admitted to hospital due to human parechovirus infections: A prospective study in Spain]. / Características epidemiológicas y clínicas de los lactantes hospitalizados por infecciones por parechovirus humanos. Estudio prospectivo en España.

INTRODUCTION: Human parechovirus (HPeV) is one of the recently described picornaviridae viruses that have been associated with fever of unknown origin (FUO), clinical sepsis, gastroenteritis, meningitis, or encephalitis in very young infants. The aim of this study is to describe the epidemiology and clinical features of these viruses. PATIENTS AND METHODS: A prospective multicentre 3-year study was conducted in 12 hospitals in Spain. Out of 850 specimens examined, 47 were positive (5.52%), with HPeV-3 being the most frequent (29 cases). Infections occurred throughout the year, but mainly in May and July, and a biennial distribution was observed. More than half (57%) were neonates, and only 2 children were older than 3 months. Fever was present in all children, with irritability in 45%, rash in 18.6%, and diarrhoea in 14%. The results of biochemical tests were all in normal range. The most common final diagnosis was FUO (61%), followed by clinical sepsis (29%). Up to 29% of infants were admitted to the intensive care unit, but only one patient had sequelae. RESULTS: Out of 850 specimens examined, 47 were positive (5.52%) for HPeV, with HPeV-3 being the most frequent (29 cases). Infections occurred throughout the year, but mainly in May and July, and a biennial distribution was observed. More than half (57%) were neonates, and only 2 children were older than 3 months. Fever was present in all children, with irritability in 45%, rash in 18.6%, and diarrhoea in 14%. The results of biochemical tests were all in normal range. The most common final diagnosis was FUO (61%), followed by clinical sepsis (29%). Up to 29% of infants were admitted to the intensive care unit, but only one patient had sequelae CONCLUSIONS: HPeV circulates in our country, mainly during spring and summer, and affects young infants with a FUO and clinical sepsis. Molecular diagnostic techniques in all hospitals could help in improving the management of patients with these infections.

First Cases of Severe Flaccid Paralysis Associated With Enterovirus D68 Infection in Spain, 2015-2016.

Enterovirus D68 was known to be the cause of mild to severe respiratory infections, but in the last few years, it has also been associated with myelitis and paralysis. This report describes the first Enterovirus D68 detections in acute flaccid paralysis cases occurring between December 2015 and March 2016 in Spain.

Molecular epidemiology of enterovirus and parechovirus infections according to patient age over a 4-year period in Spain.

The epidemiology and clinical association of enterovirus (EV) and parechovirus (HPeV) infections, as well as the type-distribution-according-to-age, were determined during a 4-year study period in Spain. During 2010-2013, a total of 21,832 clinical samples were screened for EV and the detection frequency was 6.5% (1,430). Of the total EV-negative samples, only 1,873 samples from 2011 to 2013 were available for HPeV testing. HPeV was detected in 42 (2%) of them. Positive samples were genotyped using PCR and sequencing. EV infections occurred in all age groups of patients: neonates (17%), children 28 days to 2 years (29%), children 2-14 years (40%), and adults (14%). Thirty-four different EV types were identified. HPeV infections were detected exclusively in infants <8 m (70% neonates, P < 0.05). All but one HPeV were HPeV-3. Differences in type frequency detection were found according to age and clinical manifestation. Coxsackievirus (CV)-B4 (61%), CV-B5 (83%), and HPeV-3 (64%) were more frequent in neonates than in older patients (P < 0.05). Echovirus (E)-3 (60%), E-18 (47%), E-25 (62%), CV-A6 (61%), CV-A16 (72%), and EV-71 (75%) were mainly detected in children 28 days to 2 years (P < 0.05), whereas, E-6 (79%), E-20 (88%), and E-30 (85%) were predominant in children >2 years and adults (P < 0.05). Clinically, meningitis was associated with EV (P < 0.01) whereas, encephalitis was more frequent in HPeV-infected patients. CV-B types were associated with myocarditis (90%; P < 0.05) and EV species A with hand-foot-mouth-disease/atypical exanthema (88%; P < 0.05). J. Med. Virol. 89:435-442, 2017. © 2016 Wiley Periodicals, Inc.

Molecular epidemiology of coxsackievirus B3 infection in Spain, 2004-2014.

Epidemiological and clinical characteristics of coxsackievirus B3 infections in Spain were investigated. This enterovirus (EV) type was detected mainly in young children (<6 months) and was associated with neurological (78 %) and respiratory diseases (10 %) but also with myo/pericarditis (10 %). Two myocarditis cases were fatal. Phylogenetic analysis of the VP1 region showed that genotype III circulated in the country between 2004 and 2008 and was replaced by genotype V in 2010. Furthermore, phylogenetic analysis of the 3D region indicated that recombination events have occurred and contributed to the genetic evolution of this EV type.

Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples.

Human enteroviruses (EVs) and parechoviruses (HPeVs) are important etiological agents causing infections such as meningitis, encephalitis and sepsis-like disease in neonates and young children. We have developed a real-time RT-PCR for simultaneous detection of EV and HPeV in clinical samples. Primers and probe sets were designed from the conserved 5'-noncoding region of the genomes. The sensitivity, specificity and reproducibility of the technique were measured using a set of 25 EV and 6 HPeV types. All EVs but no HPeVs were detected with the EV primers-probe set. The HPeV primers-probe set detected only the 6 HPeV types. The lower detection limit was found to be 4 and 40CCID50/ml for HPeV and EV respectively, demonstrating high sensitivity of the technique for both viruses. The threshold cycle values were highly reproducible on repeat testing of positive controls among assay runs. The assay was evaluated in 53 clinical samples of suspected meningitis, sepsis or febrile syndromes from children under 3 years. In 11 of these (21%) EVs were detected, while 4, i.e. 7.5%, were HPeV positive. Molecular typing was carried out for 73% of the viruses. In summary, the RT-PCR method developed demonstrated effectively both EV and HPeV detection, which can cause similar clinical symptoms in infants.

Molecular characterization of a coxsackievirus A24 variant that caused an outbreak of acute haemorrhagic conjunctivitis in Spain, 2004.

BACKGROUND: Coxsackievirus A24 variant is one of the major etiological agents involved in acute haemorrhagic conjunctivitis. STUDY DESIGN: An outbreak of acute hemorrhagic conjunctivitis occurred in the Southeast of Spain between September and November 2004. Cellular and molecular methods were used to identify and characterize the viral agent associated with the epidemic. RESULTS: Enterovirus was detected in the conjunctival swabs of 35 patients. None of the viruses isolated could be typed by conventional neutralization assays; however, amplification and sequencing of the 3'-end VP1 region of 19 of the samples identified coxsackievirus A24 variant as the serotype causing the outbreak. Phylogenetic analysis of the 5'-half VP1 region of the genome revealed that Spanish sequences, like other strains isolated during outbreaks of hemorrhagic conjunctivitis in American and African countries in 2003 and 2004, were closely related to the isolates detected in Korea (2002), thus proving their worldwide spread. CONCLUSIONS: This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Spain. Molecular typing in combination with phylogenetic analysis is useful to study the enterovirus epidemiology associated with epidemics.

[Enterovirus 75, a new pathogenic virus in Granada province (Spain)]. / Enterovirus 75, un nuevo virus patógeno en nuestro medio.

INTRODUCTION: Members of the genus Enterovirus are usually investigated for their etiological role in neurological syndromes. However, they are often associated with other syndromes such as febrile illness, acute respiratory infection and exanthema. In this study, clinical and epidemiological data from five subjects with infection by the recently described enterovirus 75 were analyzed in the province of Granada (Spain). METHODS: Diagnosis at the genus level was carried out by viral culture in MRC-5 and rhabdomyosarcoma cell lines. Isolate serotypes were determined by RT-PCR of a fragment of the VP1 region and subsequent sequencing of the PCR products. RESULTS: Among the five enterovirus 75 isolated, two were detected in children with aseptic meningitis (1 month and 12 years old) and three in subjects with non-neurological syndromes, i.e. acute respiratory infection, febrile illness and gastroenteritis (all were aged less than one year). The five cases were detected between December 2005 and May 2006. All patients recovered without sequelae. CONCLUSION: These data demonstrate that enterovirus 75 circulates in the south of Spain and indicate that this enterovirus serotype may be implicated in less severe non-neurological syndromes, particularly in younger children, and mainly during the cold months of the year.

Enterovirus 75, un nuevo virus patógeno en nuestro medio / Enterovirus 75, a new pathogenic virus in Granada province (Spain)

Introducción. Los miembros del género Enterovirus generalmente se investigan por su papel etiológico en procesos neurológicos. Sin embargo, a menudo se han asociado a otros síndromes, como síndrome febril, infección respiratoria aguda y enfermedad exantemática. En este trabajo hemos analizado los datos clínicos y epidemiológicos de 5 casos de infección causada por el recientemente descrito enterovirus 75 en la provincia de Granada. Métodos. El diagnóstico a nivel de género se realizó por cultivo viral en líneas celulares MRC-5 y rabdomiosarcoma (RD). El serotipo de los aislados se determinó mediante retrotranscripción-PCR (RT-PCR) de un fragmento de la región de la proteína viral 1 (VP1) y posterior secuenciación de los productos de PCR. Resultados. De los cinco enterovirus 75 aislados, 2 se detectaron en niños con meningitis aséptica (de 1 mes y 12 años de edad), y 3 en sujetos con procesos no neurológicos, que fueron infección respiratoria aguda, síndrome febril y gastroenteritis (todos menores de 1 año). Los 5 casos se detectaron entre diciembre de 2005 y mayo de 2006. Todos los pacientes se recuperaron sin secuelas. Conclusión. Estos datos demuestran la circulación de enterovirus 75 en el sur de España, e indican que este serotipo puede estar implicado en procesos no neurológicos menos graves, especialmente en niños pequeños, y sobre todo, durante los meses fríos del año (AU)

Introduction. Members of the genus Enterovirus are usually investigated for their etiological role in neurological syndromes. However, they are often associated with other syndromes such as febrile illness, acute respiratory infection and exanthema. In this study, clinical and epidemiological data from five subjects with infection by the recently described enterovirus 75 were analyzed in the province of Granada (Spain). Methods. Diagnosis at the genus level was carried out by viral culture in MRC-5 and rhabdomyosarcoma cell lines. Isolate serotypes were determined by RT-PCR of a fragment of the VP1 region and subsequent sequencing of the PCR products. Results. Among the five enterovirus 75 isolated, two were detected in children with aseptic meningitis (1 month and 12 years old) and three in subjects with non-neurological syndromes, i.e. acute respiratory infection, febrile illness and gastroenteritis (all were aged less than one year). The five cases were detected between December 2005 and May 2006. All patients recovered without sequelae. Conclusion. These data demonstrate that enterovirus 75 circulates in the south of Spain and indicate that this enterovirus serotype may be implicated in less severe non-neurological syndromes, particularly in younger children, and mainly during the cold months of the year (AU)

[Laboratory Network within the Polio Eradication Initiative (1998-2003): six years of surveillance for acute flaccid paralysis in Spain]. / Red de Laboratorios del Plan de Erradicación de la Poliomielitis (1998-2003): 6 años de vigilancia de parálisis flácida aguda en España.

INTRODUCTION: Worldwide poliomyelitis eradication was proposed in 1988 by the World Health Organization (WHO), based on reaching and maintaining high vaccination coverage and on implementing effective poliovirus infection surveillance systems. METHODS: In Spain the surveillance system focuses on active searching for acute flaccid paralysis cases through a nine-laboratory network, coordinated by the National Poliovirus Laboratory (NPL) in the National Center of Microbiology, and supported by Autonomous Community epidemiologists. Additionally, the Network sends enterovirus isolation data from other syndromes. The Laboratory Network is responsible for the primary virological study, while the NPL characterizes all polioviruses and the most epidemiologically relevant non-polio enteroviruses. RESULTS: A total of 54,533 samples were studied during the six-year period, and enteroviruses were detected in 9%. All the polioviruses isolated (n = 196), were characterized as Sabin-like (poliovirus vaccine), and among the non-polio enteroviruses, the most frequent was Echovirus 30. A total of 3% of the samples studied corresponded to the 268 acute flaccid paralysis cases or their contacts. DISCUSSION: According to the results and the WHO virological classification, Spain can be considered polio-free. However, the geographic situation of our country may facilitate the introduction of wild polioviruses that can give rise to imported poliomyelitis. Hence, the laboratory network should actively continue to participate in all the proposed WHO strategies, particularly maintenance of the poliomyelitis eradication infrastructures, and continuing monitoring and vaccination.

Red de Laboratorios del Plan de Erradicación de la Poliomielitis (1998-2003): 6 años de vigilancia de parálisis flácida aguda en España / Laboratory Network within the Polio Eradication Initiative (1998-2003): six years of surveillance for acute flaccid paralysis in Spain

Introducción. La Organización Mundial de la Salud (OMS) planteó como objetivo la erradicación de la poliomielitis en 1988, basándose fundamentalmente en alcanzar y mantener alta cobertura de inmunización e implantar sistemas eficaces de vigilancia de las infecciones por poliovirus. Métodos. En España, el sistema de vigilancia se basa en la búsqueda activa de casos de parálisis flácida aguda (PFA), mediante una red de 9 laboratorios coordinados por el Laboratorio Nacional de Poliovirus (LNP) del Centro Nacional de Microbiología y apoyados por epidemiólogos de cada comunidad autónoma. Adicionalmente, la red envía datos de aislamiento de enterovirus en cualquier síndrome clínico. En los laboratorios de la red se realiza el estudio virológico primario, mientras que en el LNP se caracterizan todos los poliovirus y los enterovirus no polio de mayor importancia epidemiológica. Resultados. El total de muestras estudiadas por la red ha sido de 54.533, con un rendimiento de enterovirus del 9%. Todos los poliovirus aislados (n = 196) se caracterizaron como Sabin-like (vacunales) y entre los enterovirus no polio, Echovirus 30 fue el dominante. El 3% de las muestras estudiadas correspondían a pacientes con PFA o sus contactos (268 casos). Discusión. España puede ser considerada como libre de polio, pero por su situación geográfica, puede ser puerta de entrada de poliovirus salvaje y dar lugar a casos importados. Por ello, debe participar activamente en todas las estrategias propuestas por la OMS, manteniendo en especial la infraestructura creada en el plan de la erradicación de la poliomielitis y continuando con la vigilancia e inmunización (AU)

Introduction. Worldwide poliomyelitis eradication was proposed in 1988 by the World Health Organization (WHO), based on reaching and maintaining high vaccination coverage and on implementing effective poliovirus infection surveillance systems. Methods. In Spain the surveillance system focuses on active searching for acute flaccid paralysis cases through a nine-laboratory network, coordinated by the National Poliovirus Laboratory (NPL) in the National Center of Microbiology, and supported by Autonomous Community epidemiologists. Additionally, the Network sends enterovirus isolation data from other syndromes. The Laboratory Network is responsible for the primary virological study, while the NPL characterizes all polioviruses and the most epidemiologically relevant non-polio enteroviruses. Results. A total of 54 533 samples were studied during the six-year period, and enteroviruses were detected in 9%. All the polioviruses isolated (n = 196), were characterized as Sabin-like (poliovirus vaccine), and among the non-polio enteroviruses, the most frequent was Echovirus 30. A total of 3% of the samples studied corresponded to the 268 acute flaccid paralysis cases or their contacts. Discussion. According to the results and the WHO virological classification, Spain can be considered polio-free. However, the geographic situation of our country may facilitate the introduction of wild polioviruses that can give rise to imported poliomyelitis. Hence, the laboratory network should actively continue to participate in all the proposed WHO strategies, particularly maintenance of the poliomyelitis eradication infrastructures, and continuing monitoring and vaccination (AU)