Global study of viral diarrhea in hospitalized children in Spain: results of structural surveillance of viral gastroenteritis net work (VIGESS-net) 2006-2008.

BACKGROUND AND OBJECTIVES: Detection and characterization of gastroenteritis cases (viruses) was conducted during eleven years through the surveillance VIGESS-net, which was created in an effort to conduct a structured surveillance of rotavirus genotypes co-circulating in Spain. STUDY DESIGN AND RESULTS: This phase includes the study of 2048 fecal samples from children <5 years old, hospitalized in fifteen different hospitals throughout Spain from March 2006 to March 2008. Of them, 821 (40.1%) samples were rotavirus positive. Rotavirus was identified as the only etiological agent in 694 (33.9%) cases and in 127 (6.2%) was found as coinfection with other enteric viruses, mainly with noroviruses. Predominant G genotypes detected were G1 (49.8%) followed by G9 (32.9%), G3 (2.6%), G8 (1.0%), G4 (0.4%), G6 (0.2%) and G12 (0.2%). The G2 was encountered in 3.2% of cases. Rotavirus mixed G-types infections occurred in 3.9% of cases. The main G/P combinations were G1P[8] (51.9%) and G9P[8] (35.6%), which predominates alternatively in the first and second period of the study. More rare combinations occur in less than 7.4% of cases. CONCLUSION: The diversity of rotavirus circulating strains suggests to maintain a surveillance system through different regions of the country.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

Identification of risk factors associated with nosocomial infection by rotavirus P4G2, in a neonatal unit of a tertiary-care hospital.

A rotavirus outbreak in newborns admitted to the 'La Paz' University Hospital, Madrid was detected, followed up and controlled. Uninfected children were selected as control subjects. Samples of faeces were taken once or twice weekly from all the newborns, including those who were asymptomatic and who were admitted to the neonatal unit for early detection of rotavirus and the positive were separated from the rest of the neonates. Contact-related precautions were taken for all patients, and alcohol solutions were used for hand washing. During the months of the outbreak, 1773 children were admitted to the hospital, 131 of whom were affected by the rotavirus infection (7.4%). Of these, 72 (55%) had symptomatic infections. In the first month of the outbreak, nine cases of necrotizing enterocolitis were diagnosed (one patient developed massive intestinal necrosis). The infections (symptomatic and asymptomatic) presented a bimodal distribution caused by a new outbreak of rotavirus type P4G2 after two patients who had acquired the infection outside the hospital were admitted when the first outbreak was subsiding. The characteristics of cases and controls were analysed using bivariate and multivariate methods (non-conditional multivariate logistic regression) to identify four risk factors strongly associated with rotavirus infection: premature birth, infections other than rotavirus, malformation, and changes in glycaemia and/or presence of jaundice.

Evaluation of two commercial enzyme immunoassays for the detection of norovirus in faecal samples from hospitalised children with sporadic acute gastroenteritis.

Two commercially available enzyme immunoassays (EIAs), IDEIA and Ridascreen, for norovirus antigen detection were evaluated with 117 faecal samples from hospitalised children with acute gastroenteritis. Eighteen of 39 samples positive by RT-PCR were characterised by sequence analysis, and 17 of these were related to norovirus genogroup II. When compared with RT-PCR, the sensitivity and specificity values were 76.9% and 85.9%, respectively, for the IDEIA assay, and 59.0% and 73.1%, respectively, for the Ridascreen assay. The sensitivity and specificity of both EIA tests require improvement, but they could both eventually be of use in the diagnosis of norovirus diarrhoea in clinical laboratories.

[Nosocomial gastroenteritis and asymptomatic rotavirus and astrovirus infection in hospitalized children]. / Gastroenteritis aguda nosocomial e infección asintomática por rotavirus y astrovirus en niños hospitalizados.

BACKGROUND: Nosocomial gastroenteritis is frequent in pediatric hospital wards. Between 20% and 50% of gastroenteritis cases caused by rotavirus and astrovirus are of nosocomial origin. OBJECTIVE: To determine the incidence of nosocomial rotavirus and astrovirus gastroenteritis in our environment, the incidence of asymptomatic infection with these viruses, and to identify the G serotypes of the rotaviruses detected. METHODS: We performed a prospective study of all children under 2 years of age admitted to a neonatology unit over a 1-year period who were followed-up for the presence of diarrhea and periodic study of feces to detect the presence of rotavirus and astrovirus antigens by enzyme immunoassay (EIA). Patients with gastroenteritis also underwent bacteria stool culture, adenovirus detection by EIA, calcivirus detection by polymerase chain reaction, and analysis of rotavirus G serotypes by EIA with monoclonal antibodies. RESULTS: Of 666 children admitted without diarrhea, 60 presented nosocomial gastroenteritis (9 % of patients admitted and 1.75 per 100 days of hospital stay): 34 presented rotavirus (5 % of patients) and two presented astrovirus (0.3 % of patients). Of the 329 patients without diarrhea who were studied, viral elimination was detected in 27: rotavirus in 23 patients and astrovirus in four. Viral infection was detected on admission in 13 patients (4 %) and after 72 hours in 14 patients (4.2 %) (asymptomatic nosocomial infection). No differences in the distribution of rotavirus G serotypes were observed between community-acquired and nosocomial gastroenteritis. CONCLUSIONS: These data confirm the importance of viral etiology in nosocomial gastroenteritis and allow us to evaluate asymptomatic fecal elimination of rotavirus as one of the factors in the transmission of this infection.

Viruses causing gastroenteritis.

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viruses are recognized as important causes 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 steadily increased. Rotavirus is the most common cause of severe diarrhea in children under 5 years of age. Astrovirus, calicivirus and enteric adenovirus are also important etiologic agents of acute gastroenteritis. Other viruses, such as toroviruses, coronaviruses, picobirnaviruses and pestiviruses, are increasingly being identified as causative agents of diarrhea. In recent years, the availability of diagnostic tests, mainly immunoassays or molecular biology techniques, has increased our understanding of this group of viruses. The future development of a safe and highly effective vaccine against rotavirus could prevent, at least, cases of severe diarrhea and reduce mortality from this disease.

First detection of group C rotavirus in children with acute diarrhea in Spain.

Group C rotavirus causes sporadic cases and outbreaks of acute diarrhea in children and adults in many countries, but has never been detected among children in Spain. In a recently conducted surveillance study to screen fecal specimens for bacteria and viruses from a cohort of 822 young children who were treated for acute diarrhea in Madrid, no pathogens were detected in fecal specimens from 238 (29%) children. In this study, we examined 147 of those specimens for group C rotavirus by EIA and PCR and found 22 (15%) were positive. Our findings demonstrate that group C rotavirus is an important cause of childhood diarrhea in Spain.

Molecular detection of human calicivirus among Spanish children with acute gastroenteritis.

A survey was conducted among Spanish children with gastroenteritis treated in an emergency room. Reverse transcription-PCR with specimens negative for other enteric pathogens was used. The minimum incidence of human calicivirus infection was 7.7%, with Lordsdale as the predominant genotype. The clinical features and severity of calicivirus and rotavirus were similar.

New immunochromatographic method for rapid detection of rotaviruses in stool samples compared with standard enzyme immunoassay and latex agglutination techniques.

Three different commercial immunologic tests for rapid detection of group A rotavirus (an immunochromatographic method, latex agglutination, and enzyme immunoassay) were used to evaluate 228 faecal specimens obtained from Spanish children with acute gastroenteritis. After resolution of 30 (13.2%) discordant results by reverse transcription-polymerase chain reaction for rotavirus, the statistical values of the enzyme immunoassay, latex agglutination, and immunochromatographic method were respectively 96%, 68%, and 99% for sensitivity; 99%, 99%, and 96% for specificity; 98%, 96%, and 92% for positive predictive value; and 98%, 88%, and 99% for negative predictive value. The immunochromatographic technique showed high sensitivity and specificity and was rapid and easy to perform in the routine clinical laboratory.

[The molecular epidemiology of the rotavirus in Spanish children. The Rotavirus Study Group (GER)]. / Epidemiología molecular de rotavirus en niños españoles. Grupo de Estudio de Rotavirus (GER).

BACKGROUND: Rotavirus are the most common etiologic agent of acute gastroenteritis in childhood. The knowledge of the circulating antigenic types is important in development of future vacunes. METHODS: Faeces from children (age < 4 years) with acute gastroenteritis admitted in the two hospitals (Hospital Severo Ochoa-Madrid and Hospital General Vic-Barcelona) have been studied prospectively during one year (October-1996 to October-1997). The detection of rotavirus was performed by ELISA (IDEIA, Dako). All samples were G-serotyping by EIA-Mabs (Silenius Laboratories) and the indeterminate or non-serotypable samples were G-genotyping by RT-PCR. P genotypes were identified by RT-PCR. RESULTS: 322 (45%) patients with acute diarrhoea causing for rotavirus were confirmed, 242 coming from the Madrid metropolitan area and other 80 from the Barcelona area. The EIA-Mabs technique made it possible to identify the G serotypes in 287 cases (89%), corresponding 207 to G1 serotype, 70 to G4 serotype and 6 to G3 serotype. In 4 patients both G1 and G4 serotypes were detected. The EIA-Mabs could not determined the serotype in 35 (11%) patients, all of whom were confirmed by RT-PCR (12 belonged to serotype G1 and 23 to serotype G4). Analysis of P genotypes was carried out in 25 patients obtained from Madrid and 17 from Barcelona; all cases were classified in the P[8] genotype. CONCLUSIONS: The most frequent serotype in both hospitals was G1. The EIA-Mabs technique were showed a high sensitivity, however, the RT-PCR technique used were even more efficient, making it possible for us to identify all the non-serotypable EIA-Mabs cases. The temporal study of circulating serotypes/genotypes of rotavirus is necessary to evaluate the efficiency of vaccines.

Ultrastructure of human astrovirus serotype 2.

The ultrastructure of human astrovirus serotype 2 (H-Ast2) grown in cell culture was analysed by electron microscopy of thin sections and negatively stained preparation. Infected LLCMK2 cells, as visualized in thin sections, contained cytoplasmic aggregates of dense or hollow-cored particles that aggregated in quasicrystalline arrays and were specifically labelled using a rabbit polyclonal anti-Ast2 antiserum. H-Ast2 particles from the supernatant of infected LLCMK2 cells in thin sections after flat- embedding were similar in size to intracellular virions. In negatively stained preparations, these virus particles had an external diameter of 41 nm and exhibited a well defined layer of surface spikes. Pentagonal and hexagonal contours were occasionally visible, and probably correspond to the projections of icosahedral structures. Star-like morphologies and particles with surface triangular hollows were seen in dark areas of the preparations only after a short treatment of the viruses of pH 10. Incubation of the viruses at pH 10.5 induced a rapid disassembly of the virus particles. The finding that the particles with icosahedral geometry and surface spikes are fully infective allows an alternative morphological model to the traditional one for astroviruses to be proposed.

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.

Characterization of a human astrovirus serotype 2 structural protein (VP26) that contains an epitope involved in virus neutralization.

An improved purification procedure for human astrovirus serotype 2 (H-Ast2) has facilitated the isolation of a neutralizing monoclonal antibody (PL-2) directed against one of the major structural proteins (VP26) of H-Ast2. A minor component (VP29) of the virus particles is also recognized by PL-2 antibody. Immunofluorescent staining indicated that VP26 (and/or VP29) has mainly a cytoplasmic location in LLCMK2-infected cells. Immunoelectron microscopy demonstrated that the PL-2 epitope was present in the surface of astrovirus particles. Pulse-chase radiolabeling and immunoprecipitation of H-Ast2-infected cell extracts identified a P86 precursor of VP26. Several intermediate protein species (P74 to P35) that shared the PL-2 epitope were also identified in the infected cells. Finally, partial N-terminal sequencing of VP29 and VP26 polypeptides demonstrated that they originated by alternative processing of P86 after residues 361 and 394, respectively. These results corroborate the location of the astrovirus structural genes at the 3' end of the viral genome included in the previously identified 2.8-kb subgenomic RNA.

Conservation of epitopes recognized by monoclonal antibodies against the separated subunits of influenza hemagglutinin among type A viruses of the same and different subtypes.

Monoclonal antibodies raised against the separated hemagglutinin subunits (HA1 and HA2) of influenza A/Vic/3/75 (H3N2) virus were tested against a large panel of human and avian strains. The epitopes recognized by most antibodies were conserved among subtype H3 viruses, but reactivity of some antibodies with members of other subtypes was also observed. Particularly, the H4 virus reacted with most antibodies directed against the HA2 subunit. These results are discussed in terms of sequence similarities between subtypes and application of these antibodies as subtyping reagents.

Isolation of cross-reactive, subtype-specific monoclonal antibodies against influenza virus HA1 and HA2 hemagglutinin subunits.

The large (HA1) and small (HA2) subunits of influenza virus A/Vict/3/75 hemagglutinin were purified in denatured form by preparative electrophoresis. Both polypeptides were used to immunize mice from which monoclonal antibodies were obtained. These antibodies reacted not only with the corresponding hemagglutinin subunit but also with purified virions. When tested by radioimmunoassay against a panel of human viruses, most anti-HA1 and -HA2 antibodies behaved as subtype-specific, whereas anti-HA antibodies, raised against purified virus, were more restricted. The anti-subunit antibodies were negative in hemagglutination-inhibition and neutralization tests. The interest of these antibodies as reagents for research and diagnosis is discussed.

An antigen-binding assay to determine the specificity of monoclonal antibodies against influenza virus and mapping of epitopes.

A simple antigen-binding assay is described for determining the specificity of monoclonal antibodies against A/Victoria/3/75 influenza virus. Monoclonal antibodies were bound to polyvinylchloride microtitre wells via protein A and anti-immunoglobulin serum. Radiolabelled cell extracts were then added and allowed to adsorb to the antigen. The bound material was eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. The method can also be used to study competitive binding of antibodies to a given antigen. In this case, one antibody was adsorbed to the wells as above and the competitor antibody was added in high excess with the radioactive antigen. The results obtained with this method are comparable to those obtained by competitive RIA or ELISA.