Etiological identification of viral agents in acute encephalitis in Guadalajara, México, 2011-2015

Introduction: Viral encephalitis is a well-known inflammatory process associated with neurological dysfunction that might derive into severe brain damage or a fatal outcome. In México there is no epidemiological data that describes the prevalence of viral agents responsible for acute encephalitis. Objective: To identify the main viral agents by real time PCR involved in acute encephalitis in Mexico. Materials and methods: We obtained cerebral spinal fluid (CSF) samples from all patients with suspected viral encephalitis admitted to the emergency service of the Hospital Civil de Guadalajara "Fray Antonio Alcalde". To identify pathogens, we performed nucleic acid extraction using real-time PCR and RT-PCR. Results: Sixty-six patients were diagnosed with acute encephalitis from 2011 to 2014. A definitive viral etiological diagnosis was established in 16 patients (24%); the main causative agents were enteroviruses in 50% of the 16 positive samples, followed by herpes simplex virus (37%) and cytomegaloviruses (12.5%). Patients with encephalitis were predominantly male (63.3%) and a seasonal predominance was observed during autumn (37.5%). The main clinical characteristics in the acute encephalitis phase were fever (48.45) and cephalea (36.3), followed by seizures, disorientation, and muscular weakness (30.3%). Kerning sign was present in two cases (3%) and other two cases presented Brudzinski's sign (3%). Conclusions: CSF PCR is a suitable diagnostic technique for the identification of viral encephalitis caused by viral infections that allows an appropriate antiviral therapeutic treatment.

Identificación etiológica de agentes virales de la encefalitis aguda en Guadalajara, México, 2011-2015 / Etiological identification of viral agents in acute encephalitis in Guadalajara, México, 2011-2015

Resumen Introducción. La encefalitis viral aguda se define como un proceso inflamatorio asociado a disfunción neurológica con desenlace fatal o daño grave permanente. En México no se han hecho estudios de identificación directa de los agentes etiológicos causales de la encefalitis viral aguda. Objetivo. Identificar mediante PCR en tiempo real los principales agentes virales causantes de encefalitis viral aguda en México. Materiales y métodos. Se obtuvo el líquido cefalorraquídeo de pacientes con sospecha de encefalitis viral que ingresaron al servicio de urgencias del Hospital Civil Fray Antonio Alcalde. Se extrajeron ácidos nucleicos para identificar los patógenos mediante PCR y PCR con transcripción inversa en tiempo real. Resultados. Se captaron un total de 66 pacientes entre el 2011 y el 2014. En 16 de los casos (24 %) se identificó el agente viral y se encontró que el principal agente causal fue el enterovirus, con ocho casos (50 %), seguido del virus del herpes simple (HSV: 37 %), con seis casos, y el citomegalovirus (CMV: 12,5 %), con dos casos. El promedio de edad fue de 25 años (0-70 años). Los casos positivos predominaron en los varones (63,3 %) y se estableció un predominio estacional en otoño (37,5 %). La mayoría de los pacientes presentó fiebre (48,4 %) o cefalea (36,3 %) y, en menor proporción, convulsiones, confusión y debilidad muscular (30,3 %) seguidas de desorientación (28,75 %) y apatía (25,7 %). En dos de los casos se observó el signo de Kerning (3 %) y en otros dos, el signo de Brudzinski (3 %). Conclusiones. La PCR en líquido cefalorraquídeo es una técnica de diagnóstico adecuada para la identificación de virus causales de encefalitis viral, lo cual permite prescribir los medicamentos específicos.

Abstract Introduction: Viral encephalitis is a well-known inflammatory process associated with neurological dysfunction that might derive into severe brain damage or a fatal outcome. In México there is no epidemiological data that describes the prevalence of viral agents responsible for acute encephalitis. Objective: To identify the main viral agents by real time PCR involved in acute encephalitis in Mexico. Materials and methods: We obtained cerebral spinal fluid (CSF) samples from all patients with suspected viral encephalitis admitted to the emergency service of the Hospital Civil de Guadalajara "Fray Antonio Alcalde". To identify pathogens, we performed nucleic acid extraction using real-time PCR and RT-PCR. Results: Sixty-six patients were diagnosed with acute encephalitis from 2011 to 2014. A definitive viral etiological diagnosis was established in 16 patients (24%); the main causative agents were enteroviruses in 50% of the 16 positive samples, followed by herpes simplex virus (37%) and cytomegaloviruses (12.5%). Patients with encephalitis were predominantly male (63.3%) and a seasonal predominance was observed during autumn (37.5%). The main clinical characteristics in the acute encephalitis phase were fever (48.45) and cephalea (36.3), followed by seizures, disorientation, and muscular weakness (30.3%). Kerning sign was present in two cases (3%) and other two cases presented Brudzinski's sign (3%). Conclusions: CSF PCR is a suitable diagnostic technique for the identification of viral encephalitis caused by viral infections that allows an appropriate antiviral therapeutic treatment.

Enterovirus y complicaciones neurológicas / Enterovirus and neurological complications

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Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes.

Identification of hotspots in the European union for the introduction of four zoonotic arboviroses by live animal trade.

Live animal trade is considered a major mode of introduction of viruses from enzootic foci into disease-free areas. Due to societal and behavioural changes, some wild animal species may nowadays be considered as pet species. The species diversity of animals involved in international trade is thus increasing. This could benefit pathogens that have a broad host range such as arboviruses. The objective of this study was to analyze the risk posed by live animal imports for the introduction, in the European Union (EU), of four arboviruses that affect human and horses: Eastern and Western equine encephalomyelitis, Venezuelan equine encephalitis and Japanese encephalitis. Importation data for a five-years period (2005-2009, extracted from the EU TRACES database), environmental data (used as a proxy for the presence of vectors) and horses and human population density data (impacting the occurrence of clinical cases) were combined to derive spatially explicit risk indicators for virus introduction and for the potential consequences of such introductions. Results showed the existence of hotspots where the introduction risk was the highest in Belgium, in the Netherlands and in the north of Italy. This risk was higher for Eastern equine encephalomyelitis (EEE) than for the three other diseases. It was mainly attributed to exotic pet species such as rodents, reptiles or cage birds, imported in small-sized containments from a wide variety of geographic origins. The increasing species and origin diversity of these animals may have in the future a strong impact on the risk of introduction of arboviruses in the EU.

[Development of a GeXP based multiplex RT-PCR assay for simultaneous detection of eight arboviruses related to encephalitis].

Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) is currently available in virus detection and defined as the simultaneous amplification of two or more DNA/RNA targets in a single reaction vessel. In this study, we attempted to modify the conventional mRT-PCR technique on a basis of GenomeLab Genetic Analysis System (GeXP). Initially, we optimized the analytical validation of the GeXP analyzer and its design of workflow and simultaneously detected eight arboviruses that related to epidemic encephalitis by verifying the specificity of mRT-PCR with Japanese encephalitis virus(JEV) cell cultures and positive strains identified previously and determining the sensitivity with in vitro-transcribed RNA of serial dilutions. The GeXP system after optimization could amplify the specific fragments related to the viruses and exposed specifically a total of 13 target genes out of eight types of arboviruses at the level of 10(2) copies/microL, and the findings suggest that the novel protocol we developed can be high-throughput and highly specific and sensitive as well as quickness in screening of the encephalitis viruses, and is promising in detection of encephalitis-associated viruses for molecular epidemiological studies.

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy.

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system.

Laboratory techniques for human viral encephalitis diagnosis.

Encephalitis is an inflammatory process involving the parenchyma of the brain. It typically presents as a clinical syndrome characterised by fever, headache and altered conscious level, often with focal neurological deficits and fits. The clinical presentation overlaps with other diseases of the central nervous system including viral and bacterial meningitis, and brain abscess. The causes of encephalitis are legion, and include principally viral but also bacterial, parasitic and fungal pathogens. Noninfectious aetiologies, especially autoimmune conditions such as potassium channel voltage gated antibodies and anti-NDMA receptor antibodies, are increasingly recognised. Diagnosis comes from clinical examination, neuroimaging and laboratory testing. With such a wide range of potential pathogens a syndromic approach to diagnosis is preferred, testing for a range of organisms. Traditional techniques such as cell culture and direct virus antigen detection have little or no role nowadays. Laboratory diagnosis of viral encephalitis is ideally based on examination of cerebrospinal fluid (CSF) for cells, protein and glucose, followed by nucleic acid amplification tests (NAAT) such as polymerase chain reaction (PCR) for a range of viral targets. Samples other than CSF sometimes give a definitive or probable aetiological diagnosis; examples include skin biopsy in rabies, and serum NAAT and antibody tests for some arboviruses and enteroviruses. Newer approaches to amplification and to multitarget detection are becoming increasingly important. Detection of intrathecal antibody production against specific viruses retains a place in diagnosis where NAAT is negative. Some of the laboratory techniques available will be discussed in this article.

Emerging viral encephalitides in dogs and cats.

Few viral pathogens resulting in encephalitis in dogs and cats have emerged over the past decade or so. All are the result of penetration through presumed species barriers and all are considered zoonoses or possible zoonotic pathogens. In all cases, encephalitis is a rare event that has low morbidity but high mortality. More viruses are likely to emerge as pathogenic in our domesticated carnivorous companions as our habitats continue to overlap with the shrinking wildlife habitats. Hopefully, however, none reach the level of distinction that was once held by rabies virus.

Detection of Israel turkey meningo-encephalitis virus from mosquito (Diptera: Culicidae) and Culicoides (Diptera: Ceratopogonidae) species and its survival in Culex pipiens and Phlebotomus papatasi (Diptera: Phlebotomidae).

Israel turkey meningo-encephalitis (ITME) virus was detected in pools of Ochlerotatus caspius Pallas and Culicoides imicola Kieffer trapped at a turkey run at Nir David during an outbreak in August 1995. Experimental membrane feeding on a blood ITME suspension showed that Culex pipiens L. became harbored virus for at least 14 d. When Phlebotomus papatasi Scopoli were fed on an infected turkey, they became infected and harbored the virus for at least 7 d. Because Phlebotomines are trapped frequently at turkey runs in Israel, they should be suspected as potential vectors of ITME.

Electrochemical immunosensor for Forest-Spring encephalitis based on protein A labeled with colloidal gold.

An electrochemical immunosensor for diagnosis of Forest-Spring encephalitis has been proposed. It comprises a screen-printed thick-film graphite electrode serving as the transducer and a layer of the Forest-Spring encephalitis antigen immobilized on the electrode and functioning as the biorecognition substance. The procedure includes formation of an antigen-antibody immune complex, localization of colloidal gold-labeled protein A on the complex, and recording of gold oxidation voltammogram, which provides information about the presence and the concentration of antibodies in blood serum. The response is proportional to the concentration of antibodies over the interval from 10(-7) to 10(-2) mg mL(-1). The detection limit is 10(-7) mg mL(-1).

Effects of immunosuppression on encephalitis virus infection in the house finch, Carpodacus mexicanus.

Immunosuppression of house finches was attempted by blood feeding Culex tarsalis Coquillett mosquitoes or by injecting birds with the corticosteroid dexamethasone or the immunosuppressant drug cyclophosphamide before and after inoculation with western equine encephalomyelitis or St. Louis encephalitis viruses. Mosquito bites (8-37 females blood feeding on each bird over a 3-d period) did not enhance the viremia response or increase the frequency of chronic infection. In contrast, dexamethasone and cyclophosphamide enhanced the amplitude and duration of the viremia response, but had no consistent effect on the antibody responses as measured by enzyme immunoassay or plaque reduction neutralization assay. Elevated viremias were followed by increases in the frequency of chronic infections with St. Louis encephalitis, but not western equine encephalomyelitis. Immunosuppression may provide a useful tool to study the chronic infection process of flaviviruses in vertebrates.

Viral encephalitis in England, 1989-1998: what did we miss?

We analyzed hospitalizations in England from April 1, 1989, to March 31, 1998, and identified approximately 700 cases, 46 fatal, from viral encephalitis that occurred during each year; most (60%) were of unknown etiology. Of cases with a diagnosis, the largest proportion was herpes simplex encephalitis. Using normal and Poisson regression, we identified six possible clusters of unknown etiology. Over 75% of hospitalizations are not reported through the routine laboratory and clinical notification systems, resulting in underdiagnosis of viral encephalitis in England. Current surveillance greatly underascertains incidence of the disease and existence of clusters; in general, outbreaks are undetected. Surveillance systems must be adapted to detect major changes in epidemiology so that timely control measures can be implemented.

Natural outbreak of viral encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax: study by nested reverse transcriptase-polymerase chain reaction.

In order to improve the sensitivity of the diagnosis of viral encephalopathy and retinopathy (VER) in sea bass, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) detection method was developed. The reverse transcription step and the first stage PCR were performed using outer primers specific for the coat protein gene, whereas a new primer set was used as inner primers for the second stage PCR. Fish were collected just before, during and after a VER outbreak occurring in a mediterranean fish farm. For each time point, ten different fish were analysed individually by nested RT-PCR, single step PCR and virus cultivation. The results showed that the frequency of positive samples was always higher using the nested RT-PCR assay. In particular, it was possible to detect nodavirus specific signals 1 month before the appearance of the first mortalities, but only by nested RT-PCR. Altogether these results showed that the sensitivity of nodavirus detection is greatly improved using a nested RT-PCR method. In particular, it was possible to monitor the presence of viral genome in asymptomatic carrier fish using this method.

Induction of nodavirus disease in seabass, Dicentrarchus labrax, using different infection models.

The aim of this study was to investigate the susceptibility of juvenile and adult seabass, which are generally thought to be refractory to nodavirus. Moreover, preliminary immunological studies were performed to examine the immune response of adult seabass. Successful transmission of the disease was experimentally demonstrated in juvenile and adult seabass as ascertained by the presence of the clinical signs of the disease, re-isolation of the virus in the SSN-1 cell line and subsequent confirmation by histopathology and immunohistochemistry. Bigger seabass not only developed the clinical disease but also suffered mortalities. Serum neutralisation titres were considered low in this study.

Viral encephalopathy and retinopathy of farmed marine fish species in Italy.

Viral encephalopathy and retinopathy, otherwise known as fish encephalitis or viral nervous necrosis (VNN), is an emerging problem in several farmed marine fish species in various geographic areas all over the world. Since summer 1995, heavy losses affecting mainly juvenile and adult sea bass (Dicentrarchus labrax) have been observed in several on-growing facilities in Italy. Dying fish show abnormal swimming behaviour and, at temperatures higher than 20-22 degrees C, mortality rates range between 15 and 50%. Neither significant external nor internal gross pathological signs, except frequent abnormal swim bladder hyperinflation, were detected. Histological investigations reveal vacuolations in the grey matter of the brain and spinal cord and in the granular layers of the retina. Serial tissue sections examined by an immunohistochemical method carried out with antisera against fish nodaviruses showed a positive reaction. Additionally, spherical virus-like particles 22-25 nm in diameter were detected by electron microscopy in negative stained preparations of brain tissues, and the same samples gave a positive reverse transcription-polymerase chain reaction (RT-PCR) for the T4 region of the fish nodavirus gene. These results indicate that both juvenile and adult sea bass subject to mass mortality in Italy since summer 1995 are infected with a fish nodavirus and strongly suggest that the identified virus is the cause of the observed mortality.

Concomitant infection with tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in patients with acute meningitis or meningoencephalitis.

From September 1992 to August 1993, 338 patients over the age of 15 years presented to the Department of Infectious Diseases, University Medical Centre Ljubljana, with acute lymphocytic meningitis. In 89 of these patients (26.3%) serum IgM and IgG antibodies against tick-borne encephalitis (TBE) virus were detected, and in 59 patients (17.5%) a borrelial etiology of disease was demonstrated by one or more of the following presence of intrathecal antibody production, seroconversion to borrelial antigens, presence of erythema migrans, and/or isolation of Borrelia burgdorferi sensu lato from skin or cerebrospinal fluid. Of the 148 patients who fulfilled criteria for TBE or borrelial infection, concomitant infection with TBE virus and B. burgdorferi sensu lato was demonstrated in 12 patients (3.6% of all patients presenting with acute lymphocytic meningitis). In the majority of patients with concomitant infection the clinical features at presentation were characteristic of, or consistent with, TBE. In addition, during follow-up studies, eight of the 12 patients subsequently developed signs and symptoms compatible with minor and/or major manifestations of Lyme borreliosis. Six patients were diagnoses with neuroborreliosis based on signs or symptoms and/or laboratory tests. These findings show that in patients with acute lymphocytic meningitis or meningoencephalitis, originating in TBE and Lyme borreliosis endemic regions, the possibility of concomitant infection should be considered.

A new tick-borne encephalitis-like virus infecting New England deer ticks, Ixodes dammini.

To determine if eastern North American Ixodes dammini, like related ticks in Eurasia, maintain tick-borne encephalitis group viruses, we analyzed ticks collected from sites where the agent of Lyme disease is zoonotic. Two viral isolates were obtained by inoculating mice with homogenates from tick salivary glands. The virus, which was described by reverse transcriptase polymerase chain reaction and direct sequencing of the amplification products, was similar to, but distinct from, Powassan virus and is provisionally named "deer tick virus." Enzootic tick-borne encephalitis group viruses accompany the agents of Lyme disease, babesiosis, and granulocytic ehrlichiosis in a Holarctic assemblage of emergent deer tick pathogens.