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

[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.

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.

Transmission networks and population turnover of echovirus 30.

Globally, echovirus 30 (E30) is one of the most frequently identified enteroviruses and a major cause of meningitis. Despite its wide distribution, little is known about its transmission networks or the dynamics of its recombination and geographical spread. To address this, we have conducted an extensive molecular epidemiology and evolutionary study of E30 isolates collected over 8 years from a geographically wide sample base (11 European countries, Asia, and Australia). 3Dpol sequences fell into several distinct phylogenetic groups, interspersed with other species B serotypes, enabling E30 isolates to be classified into 38 recombinant forms (RFs). Substitutions in VP1 and 3Dpol regions occurred predominantly at synonymous sites (ratio of nonsynonymous to synonymous substitutions, 0.05) with VP1 showing a rapid substitution rate of 8.3 x 10(-3) substitutions per site per year. Recombination frequency was tightly correlated with VP1 divergence; viruses differing by evolutionary distances of >0.1 (or 6 years divergent evolution) almost invariably (>97%) had different 3Dpol groups. Frequencies of shared 3Dpol groups additionally correlated with geographical distances, with Europe and South Asia showing turnover of entirely distinct virus populations. Population turnover of E30 was characterized by repeated cycles of emergence, dominance, and disappearance of individual RFs over periods of 3 to 5 years, although the existence and nature of evolutionary selection underlying these population replacements remain unclear. The occurrence of frequent "sporadic" recombinants embedded within VP1 groupings of other RFs and the much greater number of 3Dpol groups than separately identifiable VP1 lineages suggest frequent recombination with an external diverse reservoir of non-E30 viruses.

Aislamiento de enterovirus 75 en un paciente con un síndrome febril de origen desconocido / Isolation of Enterovirus 75 in a child with a fever syndrome of unknown origin

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First epidemic of aseptic meningitis due to echovirus type 13 among Spanish children.

Echoviruses are the commonest cause of aseptic meningitis (AM). Echovirus type 13 (EV-13) was the second enterovirus serotype associated with different local outbreaks of AM in Spain between February and October 2000. It was the first time that an epidemic AM caused by this virus was recognized in Spain. The index case appeared in the Canary Islands (Canarias). The EV-13 virus was isolated from 135 patients, predominantly from cerebrospinal fluid (CSF). All isolates were from children under 13 years. The age specific peak incidence was in infants under 1 year. Most patients had fever, headache and other meningeal signs. This enterovirus serotype, not previously detected in Spain, caused severe illness with a high attack rate.

Disfunción de marcapasos definitivo / Malfunction of permanet pacemaker

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Molecular epidemiology of echovirus 30: temporal circulation and prevalence of single lineages.

Echovirus 30 (EV30) is one of the most frequently isolated EVs, causing extensive outbreaks of EV30 aseptic meningitis in temperate climates. EV30 is antigenically heterogeneous, and three major antigenic groups have been defined, although the basis for the antigenic differences is unknown. A reverse transcription-nested PCR which amplifies the 3'-terminal region of the VP1 gene directly from clinical samples was selected for studying EV30 molecular epidemiology, since the major antigenic sites in this region reflect the serotypic pattern of this virus. The different previous approaches to the genetic classification of EV30 were analyzed. A complete study of the EV30 strains was performed by analyzing the sequences from the 112 EV30 strains amplified in this work and the complete set of EV30 strains previously published. A total of 318 strains of EV30 were divided into two broad genotypes (I and II). This classification was supported by the phylogenetic trees obtained from amino acid sequences, and it correlated with the antigenic heterogeneity of the reference strains described in earlier studies. The genotypes could be further divided into subgroups, and these subgroups could be divided into lineages based on their nucleotide distances and levels of bootstrapping. On the other hand, the subgroups and lineages did not result in the same correlation between amino acid and nucleotide differentiation. The molecular epidemiology of EV30 can be compared to influenza virus epidemiology, where prevailing lineages displace the less established lineages on the basis of immune escape. This pattern of evolution is clearly different from that of other enteroviruses. A single lineage at a time appears to be circulating worldwide. This behavior may be related to the epidemic activity of EV30.

A-549 is a suitable cell line for primary isolation of coxsackie B viruses.

A common receptor for coxsackie B virus and adenovirus has been described recently in cells of human and murine origin. Since the established cell line A-549 is suitable for adenoviruses, the potential use of A-549 cells for the isolation of coxsackie B viruses from clinical samples was investigated. All throat swabs sent to the laboratory between April 1998 and June 1999 were inoculated onto monolayers of MRC-5 and A-549 cells in tubes, and the enterovirus isolates obtained were typed. From April to June 1999, A-549 cells were compared prospectively to Buffalo green monkey (BGM) cells, considered as the most susceptible cell line for isolating coxsackie B viruses. Fifty-six out of 171 enterovirus isolates (33%) displayed a cytopathic effect (CPE) in the A-549 monolayer only, 48 isolates (28%) in the MRC-5 monolayer only, and 67 isolates (39%) in both cell lines. Most isolates that showed CPE in A-549 cells only (48 out of 56, 86%) were coxsackie B viruses, belonging to four different serotypes (B1, B2, B4, and B6). When BGM and A-549 cells were inoculated in parallel, both recovered the same number of coxsackie B isolates (n = 20), and the CPE was noted on approximately the same day. In conclusion, growth in A-549 but not MRC-5 cells identified coxsackie B viruses in most cases. A-549 was comparable to BGM for primary isolation of coxsackie B viruses.

Molecular characterization of human enteroviruses in clinical samples: comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products.

Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.

Enteroviruses in Spain: virological and epidemiological studies over 10 years (1988-97).

A total of 15,662 clinical samples were analysed for enterovirus (EV) isolation in cell cultures during a 10-year period (1988-97). Furthermore, 210 isolates of EV obtained in primary laboratories within Spain from patients with meningitis were characterized. The total number of EV typed was 758, including 727 non-polio EV and 31 Sabin-like (SL) polioviruses. Twenty-eight EV serotypes were represented. Echoviruses comprised 90% (653/727) of fully typed non-polio EV. The four most prevalent serotypes were echovirus 30, echovirus 9, echovirus 6 and echovirus 4. Echovirus 30 was the main serotype associated with meningitis. Echovirus 9 was the aetiological agent in 20 outbreaks of meningitis while the occurrence of echovirus 6 was localized in 1 year (1997). Coxsackieviruses A and B occurred in 3 and 7% of the non-polio EV respectively. Coxsackievirus B5 presented the relative greater abundance. This paper examines the epidemiology of EV in Spain to serotype level over a 10-year period with special attention to non-polio EV associated with meningitis.