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.

Prevalence of specific antibody to hepatitis E virus in the general population of the community of Madrid, Spain.

Hepatitis E virus (HEV) is an infectious agent causing hepatitis among humans. Although hepatitis E has been reported from many European countries, its incidence in Europe is largely unknown, and the prevalence of the HEV infection is also unknown for most countries of the region. Antibody to HEV (anti-HEV) was tested on 2,305 serum samples from the general population of the Community of Madrid (Spain) collected in the year 2008 among people aged 2-60 years. Total anti-HEV was tested by enzyme-immunoassay (EIA), and reactive samples were retested separately for anti-HEV IgG and IgM by recombinant immunoblot test (RIBT). Fifty samples (2.17%) displayed reactivity for total anti-HEV after EIA testing, and anti-HEV IgG was confirmed by RIBT in 25 (1.08%). The frequency of RIBT-confirmed anti-HEV ranged from 0.97% among the youngest to 3.61% among the oldest, and displayed a statistically significant trend to increasing with age. The rate of RIBT confirmation was also significantly higher among the individuals aged above 20 years old than among those younger of 21 years. HEV infection would be less frequent in the Community of Madrid than in Catalonia or the United Kingdom, and contact with HEV would be very uncommon among children and adolescents of the region. Confirmation of EIA-reactive samples by RIBT reduced the final numbers of anti-HEV testing as much as 50%, and some findings of this study suggest that such testing protocol would reflect better the real prevalence of anti-HEV in settings of low endemicity than the single testing by EIA.

Molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region.

Here we present a system for adenovirus detection and genotyping based on PCR amplification and phylogenetic analysis of a conserved hexon gene fragment. The system was validated using 157 sequences (86 previously typed and 71 clinical samples) and correctly identified species and serotype in 100% and 84% of sequences, respectively. Known associations between specific serotypes and clinical syndromes are verified. Possible new associations are described to allow further independent testing.

Rapid and sensitive diagnosis of human adenovirus infections by a generic polymerase chain reaction.

A new adenovirus specific nested polymerase chain reaction (PCR) method is described. It was designed inside the hexon protein gene of the adenovirus genome, and was able to detect DNA of all 47 human adenovirus types in a wide range of clinical samples. A sensitive internal control system able to assure proper analytical conditions for the amplification of as few as 100 molecules of a heterologous DNA was included to avoid false negative results. Sensitivity was estimated at about 10 molecules per tube of a plasmid containing an insert of the first amplification product. The method was able to detect adenovirus infection in 31/43 conjunctival scrapings from patients with acute kerato conjunctivitis 10/40 nasopharyngeal aspirates from patients admitted to hospital with acute respiratory disease and 2/26 urine samples from patients with haemorrhagic cystitis with better sensitivity than cell culture or rapid diagnosis by antigen detection by immunofluorescence (IF) in the case of respiratory specimens. Only two of 17 stools positive for a group F adenovirus specific latex immunoassay were PCR negative. The internal control system avoided a false negative result on another two stool samples. In conclusion, the method described below was shown to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection by IF.

Quantitation of polyomavirus DNA by a competitive nested polymerase chain reaction.

A new method to quantitate small amounts of DNA in clinical specimens is described. The method, a nested competitive polymerase chain reaction (ncPCR), is able to quantitate between 10 and 10(6) copies per tube of polyomavirus DNA and shows good reproducibility when clinical samples are analysed. Throughout the whole procedure, an internal standard (IS) competes for the primers with the target DNA. The internal standard, a heterologous sequence containing the four primer recognition sites, was constructed using a modification of the 'MIMIC' approach that is useful for obtaining competitor sequences for any viral, bacterial or eukaryotic target. The ncPCR method for polyomavirus was applied to cerebrospinal fluids (CSF) from AIDS patients with progressive multifocal leukoencephalopathy (PML) and urine specimens from bone marrow transplant patients affected by haemorrhagic cystitis. The results obtained suggest that the ncPCR method is a sensitive and useful method for quantitating genomic load in clinical samples.