Enterovirus asociados a Afecciones del Sistema Nervioso Central confirmados por biología molecular / Enteroviruses associated with Central Nervous System Infections confirmed by molecular biology

Aproximadamente 90% de las meningitis asépticas son causadas por enterovirus (EV), miembro de la familia de los picornavirus. Los EV son ubicuos, se diseminan por vía fecal-oral y contacto directo, responsables de brotes o casos esporádicos con importante morbilidad. El diagnóstico se basa en la presentación clínica, imágenes, estudio citoquímico del líquido cefalorraquídeo (LCR) y la Reacción en Cadena de la Polimerasa (PCR), gold estándard que reemplaza al aislamiento viral y la serología. El objetivo de este estudio descriptivo de corte transversal fue determinar la presencia de EV por RT-PCR en el LCR de pacientes con sospecha clínica de meningitis aséptica, internados en servicios públicos y privados de Asunción y departamento Central del Paraguay de noviembre de 2007 a noviembre de 2014. El material genético fue extraído mediante el mini kit ADN y ARN Qiagen® que fue sometido a RT-PCR. Se incluyeron LCR de 203 pacientes, 124 (61%) niños (4 días-15 años) y 79 (39%) adultos (16-81 años). Setenta y siete (38%) provenían de servicios públicos y 126 (62%) de privados; 115 (57%) fueron varones. Se detectó RNA de EV en 166 (82%) pacientes, 90 niños y 76 adultos, y mayor número de casos entre los meses de octubre a abril. Este es el primer trabajo en el país y muestra una importante participación del EV en pacientes por infecciones del SNC compatibles con meningitis asépticas de etiología viral. La sospecha clínica fue mayor en niños, sin embargo la proporción de resultados positivos fue mayor en adultos. Se observó mayor circulación en los meses cálidos(AU)

Meningitis aséptica en pacientes con parotiditis viral durante un brote epidémico / Aseptic meningitis associated to mumps during an epidemic outbreak

Resumen Comunicamos dos casos de meningitis aséptica asociadas a parotiditis viral en mujeres de edad mediana, una de ellas embarazada. Ambas se presentaron pocos días después del aumento de volumen parotídeo, con cefalea, fiebre y signos meníngeos, pleocitosis de predominio mononuclear en el LCR y resultados negativos para otras causas. La parotiditis fue confirmada por serología IgG e IgM positiva. Las pacientes tuvieron una evolución favorable con desaparición total de sus síntomas. Ambos casos ocurrieron durante un brote regional de parotiditis. La meningitis aséptica es una complicación frecuente de las parotiditis. Su diagnóstico puede lograrse por el aumento de volumen glandular precedente, la pleocitosis de predominio mononuclear en el LCR y una serología IgM e IgG positiva o detección genómica por RPC en muestra urinaria o salival. Esta complicación es más probable que sea observada durante brotes de parotiditis viral.

We report two cases of acute aseptic meningitis associated to mumps in middle-aged women, one pregnant. Both presented shortly after parotid gland enlargement. Neurological complications were suspected by headache, fever and meningeal signs and confirmed by CSF findings (mononuclear predominant pleocytosis) with negative results for alternative causes. Mumps were confirmed by positive IgM and IgG serology. Both patients were discharged with a favorable evolution and complete disappearance of symptoms. Cases were concurrent with a regional mumps outbreak. Conclusions: Aseptic meningitis is a rare mumps-associated neurological complication. Its diagnostic can be achieved by precedent parotid enlargement, mononuclear pleocytosis in the CSF and positive IgM and IgG serology or viral detection by PCR in urine or salivary samples. This complication would be more probably observed during mumps outbreaks.

Comparison of enterovirus detection in cerebrospinal fluid with Bacterial Meningitis Score in children / Comparação da pesquisa de enterovírus no liquor com Escore de Meningite Bacteriana em crianças

ABSTRACT Objective To measure the role of enterovirus detection in cerebrospinal fluid compared with the Bacterial Meningitis Score in children with meningitis. Methods A retrospective cohort based on analysis of medical records of pediatric patients diagnosed as meningitis, seen at a private and tertiary hospital in São Paulo, Brazil, between 2011 and 2014. Excluded were patients with critical illness, purpura, ventricular shunt or recent neurosurgery, immunosuppression, concomitant bacterial infection requiring parenteral antibiotic therapy, and those who received antibiotics 72 hours before lumbar puncture. Results The study included 503 patients. Sixty-four patients were excluded and 94 were not submitted to all tests for analysis. Of the remaining 345 patients, 7 were in the Bacterial Meningitis Group and 338 in the Aseptic Meningitis Group. There was no statistical difference between the groups. In the Bacterial Meningitis Score analysis, of the 338 patients with possible aseptic meningitis (negative cultures), 121 of them had one or more points in the Bacterial Meningitis Score, with sensitivity of 100%, specificity of 64.2%, and negative predictive value of 100%. Of the 121 patients with positive Bacterial Meningitis Score, 71% (86 patients) had a positive enterovirus detection in cerebrospinal fluid. Conclusion Enterovirus detection in cerebrospinal fluid was effective to differentiate bacterial from viral meningitis. When the test was analyzed together with the Bacterial Meningitis Score, specificity was higher when compared to Bacterial Meningitis Score alone.

RESUMO Objetivo Avaliar o papel da pesquisa de enterovírus no líquido cefalorraquidiano em comparação com o Escore de Meningite Bacteriana em crianças com meningite. Métodos Coorte retrospectiva, realizada pela análise de prontuários, incluindo pacientes pediátricos, com diagnóstico de meningite e atendidos em um hospital privado e terciário, localizado em São Paulo, entre 2011 e 2014. Foram excluídos os pacientes com doença crítica, púrpura, derivação ventricular ou neurocirurgia recente, imunossupressão, outra infecção bacteriana concomitante que necessitasse de antibioticoterapia parenteral e aqueles que receberam antibiótico 72 horas antes da punção lombar. Resultados Foram incluídos no estudo 503 pacientes. Destes, 64 foram excluídos e 94 não realizaram todos os exames para análise. Dos 345 pacientes restantes, 7 ficaram no Grupo de Meningite Bacteriana e 338 no Grupo de Meningite Asséptica. Não houve diferença estatística entre os grupos. Na análise do Escore de Meningite Bacteriana, dos 338 pacientes com possível meningite asséptica (culturas negativas), 121 deles tiveram um ou mais pontos para o Escore de Meningite Bacteriana, com valor de sensibilidade de 100%, especificidade de 64,2% e valor preditivo negativo de 100%. Dos 121 pacientes com Escore de Meningite Bacteriana positivo, 71% (86 pacientes) tiveram a pesquisa de enterovírus positiva no líquido cefalorraquidiano. Conclusão A pesquisa de enterovírus no líquido cefalorraquidiano mostrou-se eficaz em diferenciar a meningite bacteriana da viral. Analisada junto com o Escore de Meningite Bacteriana, a especificidade foi maior em comparação ao Escore de Meningite Bacteriana isolado.

Non-polio enteroviruses aseptic meningitis: embaba fever hospital admissions 2010-2011

Human enteroviruses [EV] cause a wide spectrum of both common and uncommon illnesses among all age groups. Enterically transmitted. The objective of this study was to identify non-poliovirus EV as a cause of viral aseptic meningitis [VAM] by two methods [cell culture and Real time PCR]. From October 2010 to August 2011 cerebrospinal fluid [CSF] samples were collected from 85 patients Embaba fever hospital admitted with symptoms of aseptic meningitis of any age and both sexes. The 85 CSF samples were inoculated into RD [human rhabdomyosarcoma] cell line in three blind passages to amplify isolates producing EV-like CPE. A total of 14 [16.5%] out of 85 CSF samples showed EV-like CPE. By Real time PCR 11 out of the 14 culture positive samples and 5 out of the 14 source of virus isolation original CSF were non polio EV positive. The frequency of non-polio EV meningitis hospital admissions was in the summer season [50%], spring [25%], late autumn [16.6%] and least frequency in winter [8.4%]. non-polio EV meningitis was detected in 6 out of 41 male patients [14.5%] and in 6 out of 44 female patients [13.5%]. Also non-polio EV meningitis was detected in all ages with marked increase of incidence in young children [41.6%] and old age [50%] and less in adult [8.4%]. Our data showed that the non-polioviruses EV was associated with the majority of VAM during 2010 - 2011 at the Embaba fever hospital which serves Embaba, Shoubra Elkheema, Qualyba and neighbors localities in Egypt. Rapid detection of non-polio EV meningitis is essential for making decisions about patient management and treatment

Description of a widespread outbreak of aseptic meningitis due to echovirus 30 in Rio de Janeiro state, Brazil

Echovirus 30 belongs to the genus Enterovirus and is widely associated with aseptic meningitis (AM) outbreaks. In Brazil epidemics due to this serotype were reported in several states but in Rio de Janeiro, before this study, it was only involved in sporadic episodes. We retrospectively collected data from AM notifications charts and enterovirus isolation database from Rio de Janeiro State Health Department (RJSHD) and Enterovirus Reference Laboratory in the year of 2005. An outbreak of AM was detected during March, April and May associated with a high cell culture isolation rate for echovirus 30 (17.4 percent). Male children with ages varying from 1 to 9 years were more affected. Of the 22 patients with confirmed echovirus 30 disease, clinical information was available in eight; fever, headache and vomiting were the most common manifestations. CSF analysis showed a typical pattern of viral infection with median of cellularity of 100 cells/mm³ and mononuclear cell predominance in 64.7 percent of the cases. The median of protein and glucose levels of 49 mg/dL and 56.5 mg/dL. The fatality rate was null. Despite its benign course and the lack of treatment options, aseptic meningitis surveillance is crucial for early identification of causative agents of outbreaks, which helps to avoid additional testing and inappropriate use of antimicrobials.

Echovirus 30 associated with cases of aseptic meningitis in state of Pará, Northern Brazil

Investigation of the aetiology of viral meningitis in Brazil is most often restricted to cases that occur in the Southern and Southeastern Regions; therefore, the purpose of this study is to describe the viral meningitis cases that occurred in state of Pará, Northern Brazil, from January 2005-December 2006. The detection of enterovirus (EV) in cerebrospinal fluid was performed using cell culture techniques, RT-PCR, nested PCR and nucleotide sequencing. The ages of the 91 patients ranged from < one year old to > 60 years old (median age 15.90 years). Fever (87.1 percent), headache (77.0 percent), vomiting (61.5 percent) and stiffness (61.5 percent) were the most frequent symptoms. Of 91 samples analyzed, 18 (19.8 percent) were positive for EV. Twelve were detected only by RT- PCR followed by nested PCR, whereas six were found by both cell culture and RT-PCR. From the last group, five were sequenced and classified as echovirus 30 (Echo 30). Phylogenetic analyses revealed that Echo 30 detected in Northern Brazil clustered within a unique group with a bootstrap value of 100 percent and could constitute a new subgroup (4c) according to the phylogenetic tree described by Oberste et al. (1999). This study described the first molecular characterization of Echo 30 in Brazil and this will certainly contribute to future molecular analyses involving strains detected in other regions of Brazil.

RT-PCR for confirmation of echovirus 30 isolated in Belém, Brazil

Echovirus (Echo) 30 or human enterovirus B is the most frequent enterovirus associated with meningitis cases. Epidemics and outbreaks of this disease caused by Echo 30 have occurred in several countries. In Brazil, Echo 30 has been isolated from sporadic cases and outbreaks that occurred mainly in the south and southeast regions. We used RT-PCR to examine Echo 30 isolates from meningitis cases detected from March 2002 to December 2003 in Belém, state of Pará, in northern Brazil. The patients were attended in a Basic Health Unit (State Health Secretary of Pará), where cerebrospinal fluid (CSF) was collected and stored in liquid nitrogen. Weekly visits were made by technicians from Evandro Chagas Institute to the health unit and samples were stored at -70°C in the laboratory until use. HEp-2 and RD cell lines were used for viral isolation and neutralization with specific antisera for viral identification. RNA extraction was made using Trizol reagent. The RT-PCR was made in one step, and the total mixture (50 æL) was composed of: RNA, reaction buffer, dNTP, primers, Rnase inhibitor, reverse transcriptase, Taq polymerase and water. The products were visualized in agarose gel stained with ethidium bromide, visualized under UV light. Among the 279 CSF samples examined, 30 (10.7 percent) were EV positive, 29 being Echo 30 and one was Cox B. Nineteen Echo 30 were examined with RT-PCR; 18 tested positive (762 and 494 base pairs). The use of this technique permitted viral identification in less time than usual, which benefits the patient and is of importance for public-health interventions.

Epidemiología de los enterovirus asociados a enfermedades neurológicas / Epidemiology of enterovirus associated with neurologic diseases

El presente estudio describe los resultados de la investigación de los enterovirus humanos (HEV) mediante cultivo celular y reacción en cadena de la polimerasa y su tipificación molecular en 2167 casos de parálisis fláccida aguda, meningitis aséptica y encefalitis aguda, obtenidos entre 1991 y 1998 en la Argentina. La frecuencia de detección de HEV en parálisis fláccida aguda fue 19.5% (130/666) y de poliovirus Sabin 5.4% (36/666). La tasa de detección de HEV en los casos de meningitis fue 28.8% (231/801) y en encefalitis 3.0% (21/700). El grupo etario más afectado por las meningitis fue entre 1 y 9 años (75.3%) y en los casos de parálisis fláccida aguda, de 1 a 4 años (58%). En muestras de brotes de meningitis se identificó echovirus (E) 4, E9, E30 y E17, y en casos esporádicos virus coxsackie A (CAV) 2, B (CBV) 2 y CBV5, E7, E11, E19, E24 y E29, y enterovirus (EV) 71. Finalmente, en casos de encefalitis se detectó E4, E7 y E24. En casos de parálisis fláccida aguda se identificaron 28 serotipos distintos de enterovirus no polio. En la Argentina y en otros países latinoamericanos existe escasa información acerca de la circulación de los HEV y su relación con diversas enfermedades neurológicas. Este estudio proporciona información que puede servir como base para posteriores investigaciones.

This report describes the results of human enterovirus (HEV) detection and characterization using cell culture, polymerase chain reaction and molecular typing in 2167 samples obtained from acute flaccid paralysis, aseptic meningitis and acute encephalitis patients, from 1991 to 1998 in Argentina. HEV were isolated in 130 out of 666 cases (19.5%) and 36 out of 666 (5.4%). HEV RNA was detected in 28.8% (231/801) and 3.0% (21/700) of the patients with meningitis and encephalitis, respectively. Children with ages ranging from 1 to 9 years accounted for 75.3% of the meningitis cases and from 1 to 4 years for 58% of acute flaccid paralysis patients. Echovirus 4 (E4), E9, E30 and E17 were identified from meningitis outbreaks. Coxsackievirus A2 (CAV2), CBV2, CBV5, E7, E11, E19, E24, E29 and enterovirus 71 were recovered only from sporadic cases. Three different serotypes were identified in encephalitis patients: E4, E7 and E24. A total of 28 different serotypes of non-polio enteroviruses were detected from acute flaccid paralysis cases. The information here presented contributes to improving our knowledge about enteroviruses epidemiology in Argentina and their relationship with different neurological diseases. This study provides valuable data that could be useful to further research.

Pesquisa de enterovírus em casos de síndrome de meningite asséptica de Belém, PA / Investigation of enterovirus in cases of aseptic meningitis syndrome of Belém, PA

Com o objetivo de isolar e identificar os sorotipos de enterovírus, agentes etiológicos mais freqüentes da síndrome de meningite asséptica, foram estudadas amostras de líquor de pacientes da unidade de saúde de referência da Cidade de Belém-PA, do período de março de 2002 a março de 2003. As amostras foram inoculadas em cultivos celulares RD e HEp-2, e as positivas identificadas por neutralização ou imunofluorescência indireta. De 249 amostras, 33 (13,2 por cento) foram positivas sendo 57,6 por cento (n=19) em pacientes menores de 11 anos (p<0,03) e predominantemente (72,7 por cento) naqueles do sexo masculino (p<0,008). Os sorotipos isolados foram: Echovírus 30 (n=31), Coxsackievírus B5 (n=1) e Echovírus 30 e 4. Em conclusão, estudos deste tipo servem também para melhor compor o quadro nacional, ainda pouco definido, sobre os agentes enterovirais mais prevalentes em casos de SMA.

Detección de enterovirus mediante transcripción reversa y reacción de polimerasa en cadena en líquido cafalorraquídeo de niños con meningitis aséptica / Detection of enterovirus by reverse transcriptase and polymerase chain reaction in cerebrospinal fluid samples of children with aseptic meningitis

Los enterovirus causan cerca de 90 por ciento de las meningitis asépticas (MA). La ausencia de un buen método de diagnótico etiológico determina con frecuencia tratamientos innecesarios. En este estudio se comparó la detección de enterovirus en líquido cefalorraquídeo (LCR) usando cultivos celulares e inmunofluorescencia indirecta (CC/IFI), con la reacción de polimerasa en cadena (RT-PCR). Se estudiaron 22 muestras de LCR provenientes de niños con MA (casos) y 17 de niños con otras patologías (controles). Todos fueron atendidos en servicios pediátricos del Hospital Carlos Van Buren de Valparaiso. Hubo 100 por ciento de concordancia entre ambos métodos en los 22 niños con MA, encontrándose 21 casos positivos para enterovirus. Entre los 17 controles, 2 muestras resultaron por RT-PCR y una de ellas lo fue también por CC-IFI. La RT-PCR resultó un método sencible y específico. Considerando la rapidez con que se puede tener los resultados es esperable que su uso rutinario sea beneficioso para los pacientes con MA

RT-nested PCR for the detection of enterovirus in biological samples from patients with suspected enteroviral infections

In this study, we have tested a reverse transcription (RT) nested polymerase chain reaction (nPCR) for detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF), serum samples, and conjunctival swabs (CS) from patients with suspected enterovirus infections. A specific 113-bp fragment was amplified using primers designed based on 5' non coding region of the enterovirus genome. The enterovirus RT-nPCR was able to detect 0.001 plaque forming unit (pfu)/ml. Since no PCR product was detected in each of the CSF, CS and serum samples from patients with proven-non-enterovirus viral infections, this method was found to be specific. EV RNA was detected in all 30 culture-confirmed CSF samples and yielded positive results in 5 out of 7 additional cases of culture-negative CSF samples with other evidences of enterovirus infection. Overall, EV RNA was detected in 95 of the patients with clinical diagnosis of viral central nervous system (CNS) disease and confirmed enterovirus infection. Furthermore, we were able to detect EV RNA in 24 (47) out of 51 CSF samples from patients with clinical diagnosis of viral CNS disease and negative laboratory evidence of viral infection. The percentage of positive EV RNA detection in paired CSF and serum samples from 11 patients with an enterovirus isolate in CSF was 100 (11 of 11) and 73 (8 of 11), respectively. In addition, EV-specific IgM was detected in 64 (7 of 11) of the sera tested. The method was also tested against 136 samples of CS from patients with clinical diagnosis of acute hemorrhagic conjunctivitis. Ninety nine of them resulted positive (73), while only 27 (20) had been positive for viral culture. In summary, our study shows the importance of enterovirus RT-nPCR for the diagnosis of enterovirus associated disease in different kind of biological samples and different types of diseases.

Caracterización etiológica de las meningoencefalitis asépticas en la provincia Ciego de Avila, Cuba / Etiologic characterization of aseptic meningoencephalitis in the province of Ciego de Avila, Cuba

Se realiza análisis prospectivo aleatorio a 198 pacientes con meningoencefalitis aséptica (MEA) ingresados en el servicio de medicina del hospital provincial ®Dr. Antonio Luaces Iraola¼ de Ciego de Avila, Cuba. Se les efectuó estudios con sueros pareados para determinar anticuerpos neutralizantes a enterovirus, pruebas de hemoglutinación, neutralización viral para arbovirus, hemolítica para leptospira y estudio virológico de heces fecales buscando enterovirus. En 36.8 por ciento de los pacientes se encontró como agente etiológico un enterovirus. El segundo agente en frecuencia fue la leptospira en 14.6 por ciento. Se determinó el agente etiológico en 51.4 por ciento de los pacientes estudiados

Molecular biological characterization of enterovirus variant isolated from patients with aseptic meningitis

In Korea, there was a big outbreak of aseptic meningitis in 1993. Six clinical isolates of enterovirus were obtained from patients with aseptic meningitis and were identified as echovirus type 9 by serotyping with a pool of neutralizing antisera. For molecular characterization of the isolates, the nucleotide sequences of 5'-noncoding region (NCR), VP4, VP2, VP1, 2A and 2C regions of the isolates were compared with the corresponding regions of echovirus type 9 Hill and Barty strains. Unlike Hill strain, Barty strain contained a C-terminal extension to the capsid protein VP1 with an RGD (argnine-glycine-aspartic acid) motif. To determine whether similar structural features were present in our isolates, their nucleotide sequences including the VP1 region were analyzed. All isolates exhibited the VP1 extension with the RGD motif. We concluded the Korean isolates in the year of 1993 as the echovirus type 9 Barty strain although the isolates showed 15-20% nucleotide sequence differences in the several genomic regions.

Acute aseptic meningitis and encephalitis with viral etiology in children from Tehran

The incidence of aseptic meningitis and encephalitis in relation to neurotropic viruses was studied in 430 children <14 years of age, from September 1992 to October 1996 in Tehran. Enterovirus was isolated from CSF in 15% of patients with acute aseptic meningitis and all isolates were typed as coxsackievirus types B5 [95%] and B4 [5%]. The highest incidence of enteroviral meningitis [42.5%] was in children under 12 months of age. We found an incidence of meningitis and meningoencephalitis due to mumps-virus [48%] in children under 14 years old. The incidence peak was 5-9 year old in children. The overall rate of infection in boys was three times higher than that in girls. The highest rates of enteroviral meningitis and mumps-virus meningitis and encephalitis were 50% in the summer and 53.5% in the spring [p<0.001], respectively. In this study, Herpes simplex virus type-1, Varicella Zoster virus and measles virus were determined to be the etiologic viral agents of acute encephalitis in 6 children<10 years of age. Our study indicates that mumps virus and enterovirus are important causative agents of aseptic meningitis and meningoencephalitis among children in Tehran