Monitoring of children with acute gastroenteritis in Madrid, Spain, during 2010-2011: rotavirus genotype distribution after the vaccines introduction.

BACKGROUND AND OBJECTIVES: A structured surveillance study was conducted on children with diarrhea who were hospitalized in Madrid (Spain) during 2010-2011, in order to describe temporal, geographic, and age-related trends in rotavirus (RV) strains after the introduction of the RV vaccines in our country. STUDY DESIGN AND RESULTS: A total of 370 children were enrolled, with RV being detected in 117 (31.6%) cases. Coinfections were detected mainly with rotavirus, astrovirus and norovirus. The most prevalent rotavirus G type was G1 (60.7%) followed by G2 (16.09%), G9 (5.9%), and G12 (5.1%). The G12 genotype appeared for the first time in 2008 in Spain, and it has increased to 5.1% of the cases in this report. Some uncommon P genotypes, such as P[14] and P[6], both with a low percentage, were found. The samples with G1 G2, G9 and G12 genotypes appeared in all ages, but were significantly higher in children under 2 years old. CONCLUSION: A long-term structured surveillance is required in the Spanish post vaccine era, in order to determine the prevalence and variability of RV genotypes. This will especially be needed to distinguish between changes occurring as a result of natural fluctuation in genotype or those (changes) that could be mediated by population immunity to the vaccines. In addition, it will be necessary to study the impact of the current vaccines on the circulating rotavirus strains and on the overall reduction in the prevalence of rotavirus disease among children in Spain.

[Microbiological profile of acute viral gastroenteritis attended in a paediatric department of an area with high vaccine cover against rotavirus]. / Caracterización microbiológica de las gastroenteritis agudas virales atendidas en un servicio de pediatría en un área de alta cobertura vacunal frente a rotavirus.

INTRODUCTION: Vaccination against rotavirus has led to a significant decline of the disease. The aim of the present work is to evaluate the clinical and epidemiological features of the viral acute gastroenteritis (AGE) in an area with high immunization coverage against rotavirus. METHOD: A prospective microbiological evaluation was made of stool culture and Real Time Polymerase Chain Reaction (RT-PCR) to gastroenteric virus and genotyping of rotavirus strains in < 5 year-old with AGE episodes attended by or admitted to our hospital from November-March of 2009-2010 and 2010-2011. RESULTS: A total of 51 patients were included, with a mean age (standard deviation) of 19.1 (13.9) months. Stool culture was negative in 23 samples (45% of the samples analyzed), and it was identified a responsible microorganism in 70% by the RT-PCR (16 samples). Rotavirus was the most common isolated microorganism (53%), and G1[P8] the most frequent genotype. A co-infection was detected in 14% of samples (7 patients), and rotavirus and astrovirus were the most frequent etiological agents involved. CONCLUSIONS: Rotavirus, basically G1[P8], is the most common AGE responsible agent identified in our study. The use of RT-PCR enhances the AGE diagnostic sensitivity, and uncovers an important number of viral co-infections.

Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus.

Human astrovirus (HAstV) has been recognized as the second most common cause of diarrhoea among children under 5 years old. To date, the true incidence of HAstV was underestimated when using enzyme immunoabsorbent assays (EIAs) and conventional reverse transcription (RT)-polymerase chain reaction (PCR) methods. The sensitivity of detection of EIA is insufficient and, although RT-PCR is more sensitive than EIA, the time required is a limitation for astrovirus detection. The aim of the study was to develop a real-time RT-PCR method in order to increase the sensitivity, to quantify the viral load and to minimize the time required for HAstV detection. The real-time RT-PCR reported here requires only one rapid step to obtain a high sensitivity (0.0052 infectious units (IU) (0.0026 IU/microl)) in all human astrovirus detected. The real-time RT-PCR detected IUs down to a 10(-6) dilution with an improvement in the detection limit of factor 10(4), whereas the conventional RT-PCR detected down to IUs 10(-2) dilution. This process is able to reduce the time of the assay and avoids the risk of contamination. The method described below has been validated with a panel of 100 clinical samples and the results obtained confirmed the high specificity of the assay; consequently, the application of this assay for molecular diagnosis is feasible as a versatile tool for ascertaining the true implication of HAstV in acute viral gastroenteritis.

De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody.

Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent.

Norovirus-associated encephalitis in a previously healthy 2-year-old girl.

We describe the case of 2-year-old girl with encephalitis associated with norovirus infection. The viral genome was detected in cerebrospinal fluid and stool by reverse transcription polymerase chain reaction. This is the first time in Spain and the second worldwide that the genome of norovirus from the stool sample and 2 cerebrospinal fluid samples were genetically characterized and matched.

Astrovirus acute gastroenteritis among children in Madrid, Spain.

BACKGROUND: Human astroviruses cause infantile gastroenteritis worldwide, but the prevalence of disease varies greatly by setting. Since 1997 we have conducted a survey to determine the causes of diarrhea among Spanish children attending an emergency room in Madrid and to characterize the clinical features of viral-associated gastroenteritis. OBJECTIVES: To define the epidemiologic role of astrovirus-associated gastroenteritis in Spanish children, to review its clinical features and to compare these illnesses with those caused by rotavirus. To assess the sensitivity of two methods of detection [enzyme-linked immunosorbent assay (EIA) and reverse transcriptase (RT)-PCR]. METHODS: Fecal specimens from 822 children with acute diarrhea treated at an emergency room were screened by EIA assays. Random astrovirus-positive samples were characterized by RT-PCR and nucleotide sequencing for their phylogenetic grouping. RESULTS: Astrovirus was detected in 44 (5.3%) of 822 specimens tested by EIA. No pathogens were detected in fecal specimens from 238 (29%) children; however, in 137 of those with adequate remaining specimens, we found an additional 50 (6.1%) that were positive by RT-PCR. HAstV-1 was the most prevalent type followed by HAstV-2. The gastroenteritis associated with astrovirus alone was slightly less severe and had a lower score or risk of hospitalization than that associated with rotavirus (P < 0.05). CONCLUSIONS: Astrovirus was found in 11.4% of all children whom we tested for enteric viral and bacterial pathogens, making it the second most common cause of acute gastroenteritis among Spanish children. True prevalence of astrovirus could be underestimated if only EIAs were used for detection.

Acute viral gastroenteritis: proportion and clinical relevance of multiple infections in Spanish children.

Dual infections associated with acute infectious diarrhoea and its microbiological, epidemiological and clinical findings have been evaluated in patients selected from a comprehensive survey of children under 4 years old, admitted to hospital emergency rooms from October 1996 to November 1997. A total of 820 children (433 males and 387 females) were enrolled. Stools were tested for rotavirus, adenovirus, astrovirus and bacterial enteropathogens. Patients were grouped according to age, and the seasonality of mixed infections was evaluated. Clinical trends and severity of gastrointestinal disease by Ruuska's score were also analysed. Mixed infections were identified in 39 cases (5 %), of which 23 were males and 16 were females. The majority of cases were in the 7-18-month age group (26 cases) and occurred in autumn (67 %). Virus-virus co-infections were more frequent (26/39) than virus-bacteria co-infections (13/39). More than two infectious agents were detected in only four cases. The most common viral co-infections were rotavirus-astrovirus (13/26) and rotavirus-adenovirus (10/26). The present report is the first prospective analysis of clinical-epidemiological trends of dual infections in young Spanish children with acute viral gastroenteritis. Our results emphasize the clinical importance of mixed infections as a cause of severe diarrhoea in children.

Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form.

The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.

Nosocomial rotavirus gastroenteritis in Spain: a multicenter prospective study.

BACKGROUND: The objective of this study was to assess the incidence of nosocomial rotavirus gastroenteritis among children <2 years of age. METHODS: We conducted a prospective active surveillance for acute gastroenteritis (AGE) in the pediatric wards of 3 representative hospitals in Valencia (Spain) from October 2006 to March 2007, among children between 1 and 23 months of age with acute diarrhea. Children were followed up for 3 days after discharge. We obtained clinical and demographic information from participants and tested their stool specimens for rotavirus. RESULTS: A total of 1576 children were hospitalized at the 3 hospitals and 1300 (82.5%) were followed up as the study cohort. In 69 children, AGE started 48 hours after admission and were considered nosocomial infections. In 35 of the 59 cases where stool samples were obtained, rotavirus (RV) was present (59%), and in 12 of them symptoms started after discharge. The accumulated incidence of nosocomial rotavirus disease during the study period was 2.8 cases per 100 inpatients (95% CI: 1.9-3.8), and the incidence rate was 4.8 cases per 1000 hospital days (95% CI: 3.2-6.5). The most commonly found genotype in nosocomial infection was G9P[8], in 23 cases (66%), followed by G1P[8] in 4 cases (11%). The total economic cost was 883 euro per case. CONCLUSION: Active surveillance demonstrated that the burden of nosocomial rotavirus disease is substantial, and G9P [8] was the genotype found most frequently. Following up children after discharge from hospital allowed the discovery of cases of nosocomial RVAGE which are missed in most other studies.

[Detection of a rotavirus G9P[8] outbreak causing gastroenteritis in a geriatric nursing home]. / Detección de rotavirus G9P[8] como agente causante de un brote de gastroenteritis en una residencia de ancianos.

INTRODUCTION: The aim of this study was to describe an outbreak of gastroenteritis due to rotavirus in a geriatric nursing home. METHOD: Stool samples from 8 patients were studied. Antigen detection was carried out by ELISA, and molecular typing was performed by RT-PCR. RESULTS: Rotavirus antigen was detected in 6 patients. Typing demonstrated a common genotype (G9P[8]). CONCLUSION: The outbreak detected in elderly persons suggests a loss of immunity with age or a lack of protection against an emergent genotype.

Prevalence and clinical characteristics of norovirus gastroenteritis among hospitalized children in Spain.

BACKGROUND: The importance of norovirus as a cause of gastroenteritis outbreaks is well documented, but the role of norovirus in sporadic acute severe gastroenteritis is not so well established. The aim of this study was to determine the prevalence and clinical characteristics of norovirus gastroenteritis among hospitalized children. METHODS: A prospective study was conducted in children less than 5 years old, admitted with acute gastroenteritis between January 2005 and January 2008 to the Pediatrics Department of the Universitary Hospital, Albacete, Spain. Demographic and clinical data were collected. A stool sample from each child was screened for enteropathogenic bacteria and tested by reverse transcription polymerase chain reaction for rotavirus, astrovirus, norovirus, and sapovirus and by immunochromatographic method for enteric adenoviruses. RESULTS: Norovirus was the second most frequent pathogen after rotavirus, being detected in 61 (17.3%) of the 352 children enrolled, in 29 of them (8.2%) as single agent. Mixed infections involving other viruses or bacteria were present in 52.4% of norovirus positive samples, a nosocomial source of infection was demonstrated in 17.2%. Norovirus infection was more prevalent in winter and affected mainly children less than 2 years of age. Vomiting was present in 68% and fever in 48.3% of cases, 3 children had nonfebrile seizures. Compared with rotavirus enteritis, norovirus infection was slightly less severe (in terms of severity score and need of intravenous rehydration) and fever was less frequent. CONCLUSIONS: Norovirus was a frequent cause of acute severe sporadic gastroenteritis in children representing the second etiologic agent after rotavirus.

Monitoring of children with acute gastroenteritis in Madrid, Spain, during 2010–2011: Rotavirus genotype distribution after the vaccines introduction

BACKGROUND AND OBJECTIVES: A structured surveillance study was conducted on children with diarrhea who were hospitalized in Madrid (Spain) during 2010-2011, in order to describe temporal, geographic, and age-related trends in rotavirus (RV) strains after the introduction of the RV vaccines in our country. STUDY DESIGN AND RESULTS: A total of 370 children were enrolled, with RV being detected in 117 (31.6%) cases. Coinfections were detected mainly with rotavirus, astrovirus and norovirus. The most prevalent rotavirus G type was G1 (60.7%) followed by G2 (16.09%), G9 (5.9%), and G12 (5.1%). The G12 genotype appeared for the first time in 2008 in Spain, and it has increased to 5.1% of the cases in this report. Some uncommon P genotypes, such as P[14] and P[6], both with a low percentage, were found. The samples with G1 G2, G9 and G12 genotypes appeared in all ages, but were significantly higher in children under 2 years old. CONCLUSIÓN: A long-term structured surveillance is required in the Spanish post vaccine era, in order to determine the prevalence and variability of RV genotypes. This will especially be needed to distinguish between changes occurring as a result of natural fluctuation in genotype or those (changes) that could be mediated by population immunity to the vaccines. In addition, it will be necessary to study the impact of the current vaccines on the circulating rotavirus strains and on the overall reduction in the prevalence of rotavirus disease among children in Spain

INTRODUCCIÓN: durante el período 2010-2011 se llevó a cabo un estudio de vigilancia entre los niños con diarrea que fueron hospitalizados en Madrid (España), con el fin de describir los genotipos circulantes de rotavirus (RV) después de la introducción de las vacunas en nuestro país. MÉTODO Y RESULTADOS: Un total de 370 niños fueron incluidos en el estudio. RV se detectó en 117 (31.6%)casos. Las coinfecciones detectadas fueron rotavirus, astrovirus y norovirus. El genotipo más prevalente fue G1 (60.7%) seguido de G2 (16.09%), G9 (5.9%) y G12 (5.1%). G12 apareció por primera vez en 2008 en España y ha aumentado hasta el 5.1% de los casos en este estudio. Algunos genotipos P infrecuentes como P[14] y P[6], fueron identificados, ambos con un porcentaje bajo. G1,G2, G9 y G12 se aislaron en todas las edades, pero son significativamente más frecuentes en los niños menores de 2 años de edad. CONCLUSIÓN: Con el fin de conocer la prevalencia y variabilidad de los genotipos de RV, la vigilancia a largo plazo será necesaria en la era postvacunal. Esta será especialmente necesaria para distinguir entre cambios que se producen como resultado de la fluctuación natural del genotipo o los cambios que podrían estar mediados por inmunidad de la población a las vacunas. Además, será necesario estudiar el impacto de las vacunas actuales sobre las cepas de rotavirus circulantes y en la reducción global de la prevalencia de la enfermedad por rotavirus en nuestro país

Caracterización microbiológica de las gastroenteritis agudas virales atendidas en un servicio de pediatría en un área de alta cobertura vacunal frente a rotavirus / Microbiological profile of acute viral gastroenteritis attended in a paediatric department of an area with high vaccine cover against rotavirus

INTRODUCCIÓN: La vacunación frente a rotavirus ha condicionado un descenso significativo de la enfermedad. El presente trabajo pretende evaluar las características clínicas y epidemiológicas de la gastroenteritis aguda (GEA) por virus en un área de alta cobertura vacunal frente a rotavirus. Método Evaluación prospectiva microbiológica mediante coprocultivo y reacción en cadena de la polimerasa en tiempo real (RT-PCR) para virus gastroentéricos, y genotipificado de las cepas de rotavirus de los casos de GEA en menores de 5 años que acudieron a urgencias o fueron hospitalizados en nuestro centro de noviembre a marzo de 2009-2010 y 2010-2011.ResultadosSe incluyeron 51 pacientes con una edad media (desviación estándar) de 19,1 (13,9) meses. El coprocultivo fue negativo en 23 muestras (45% de los casos), identificándose mediante RT-PCR un microorganismo causal en 16 de estas muestras (el 70%). El rotavirus fue el microorganismo más detectado (53%) y el genotipo G1[P8] el más abundante. En el 14% de los casos (7 pacientes) se identificó coinfección, siendo el rotavirus y el astrovirus los agentes más frecuentemente involucrados. Conclusiones El rotavirus, principalmente el G1[P8], se ha identificado como la causa más frecuente de GEA en nuestro estudio. La utilización de RT-PCR mejora significativamente la sensibilidad diagnóstica en el contexto de la GEA, y pone de relieve un porcentaje elevado de coinfecciones virales

INTRODUCTION: Vaccination against rotavirus has led to a significant decline of the disease. The aim of the present work is to evaluate the clinical and epidemiological features of the viral acute gastroenteritis(AGE) in an area with high immunization coverage against rotavirus. METHOD: A prospective microbiological evaluation was made of stool culture and Real Time Polymerase Chain Reaction (RT-PCR) to gastroenteric virus and genotyping of rotavirus strains in < 5 year-old with AGE episodes attended by or admitted to our hospital from November-March of 2009-2010 and 2010-2011.RESULTS: A total of 51 patients were included, with a mean age (standard deviation) of 19.1 (13.9) months. Stool culture was negative in 23 samples (45% of the samples analyzed), and it was identified a responsible microorganism in 70% by the RT-PCR (16 samples). Rotavirus was the most common isolated microorganism (53%), and G1[P8] the most frequent genotype. A co-infection was detected in 14% of samples(7 patients), and rotavirus and astrovirus were the most frequent etiological agents involved CONCLUSIONS: Rotavirus, basically G1[P8], is the most common AGE responsible agent identified in our study. The use of RT-PCR enhances the AGE diagnostic sensitivity, and uncovers an important number of viral co-infections

[Rotavirus and other viruses causing acute childhood gastroenteritis]. / Rotavirus y otros virus productores de gastroenteritis aguda en la infancia.

Acute gastroenteritis is one of the most common diseases, affecting children worldwide. Viruses are recognized as a major cause of this disease, particularly in children. Since the Norwalk virus was identified as a cause of gastroenteritis, the number of viral agents associated with diarrheal disease in humans has progressively increased. Rotavirus is the most common cause of severe diarrhea in children under 5 years of age. Human astroviruses, caliciviruses and enteric adenovirus are also important etiologic agents of acute gastroenteritis. Other viruses such as toroviruses, coronaviruses, picobirnaviruses, Aichi virus and human bocavirus are increasingly being identified as causative agents of diarrhea. Vaccination against rotavirus could prevent cases of severe diarrhea and reduce the mortality attributable to this disease.

European multicenter evaluation of commercial enzyme immunoassays for detecting norovirus antigen in fecal samples.

A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.

[Assessment of two methods of antigenic detection by ELISA for the diagnosis of norovirus outbreaks]. / Evaluación de dos métodos de detección antigénica por ELISA para el diagnóstico de brotes causados por norovirus.

INTRODUCTION: The aim of this study was to assess two ELISA techniques for the detection of outbreaks of norovirus. METHOD: One-hundred and sixty-five fecal samples from patients affected in 30 outbreaks were studied. RESULTS: On the basis of a specific consensus criterion between techniques for confirming outbreaks, the sensitivity and specificity was respectively 80% and 90% for RIDASCREEN, and 80% and 100% for IDEIA. CONCLUSION: These techniques could be useful for rapid detection of norovirus outbreaks in laboratories that lack molecular confirmation techniques.

Human rotavirus G9 and G3 as major cause of diarrhea in hospitalized children, Spain.

In Spain, diarrhea remains a major cause of illness among infants and young children. To determine the prevalence of rotavirus genotypes and temporal and geographic differences in strain distribution, a structured surveillance study of hospitalized children <5 years of age with diarrhea was initiated in different regions of Spain during 2005. Rotavirus was detected alone in samples from 362 (55.2%) samples and as a coinfection with other viruses in 41 samples (6.3%). Enteropathogenic bacterial agents were detected in 4.9% of samples; astrovirus and norovirus RNA was detected in 3.2% and 12.0% samples, respectively; and adenovirus antigen was detected in 1.8% samples. Including mixed infections, the most predominant G type was G9 (50.6%), followed by G3 (33.0%) and G1 (20.2%). Infection with multiple rotavirus strains was detected in >11.4% of the samples studied during 2005.

Vaccinia virus recombinant expressing an 87-kilodalton polyprotein that is sufficient to form astrovirus-like particles.

Human astrovirus is an important cause of acute gastroenteritis. We have generated, for the first time, a vaccinia virus recombinant expressing the astrovirus 87-kDa structural polyprotein. The results demonstrate that this expression results in the formation of virus-like particles in the absence of other astrovirus proteins and genomic RNA. The purified trypsin-activated virus-like particles strongly resemble the complete astrovirus particles.

Diversity of group A human rotavirus types circulating over a 4-year period in Madrid, Spain.

The incidence and distribution of human rotavirus G types among children under 5 years old with acute gastroenteritis were determined over a 4-year period (1998 to 2002) by using monoclonal antibodies and reverse transcription-PCR methods. Rotavirus was detected in 1,155 (31%) of 3,760 specimens tested. Rotavirus was studied in every month of the 48-month survey period. Rotavirus activity occurred mainly (51%) in the typically cooler months in Spain (November to February). The age distribution of rotavirus-positive cases showed that 90% of patients (1,038 of 1,155) were under 2 years old. Rotavirus types were determined for 576 of 1,155 patients (50%). G1 was the main genotype detected (53%), and the second most common was G4 (24%). The G2, G9, and G3 rotavirus types were detected in 14, 6, and 2% of the cases, respectively. Dual infections were detected in only 0.6%. The seasonal distribution of genotypes showed a significant genotypic shift: whereas G4 strains predominated (57%) during the 1998 to 2000 seasons, the G1 gradually increased to account for 75% in the 2000 to 2002 seasons. In addition, the present study reports the first detection of the G9 genotype in human fecal samples in Spain. Therefore, additional types may be required for vaccine development strategies that currently target only types G1 to G4.