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.

Brote epidémico de meningitis viral causado por echovirus tipo 4 en la provincia de Misiones / Outbreak of viral meningitis caused by echovirus type 4 in Misiones province

Se realizó un estudio retrospectivo a fin de describir un brote epidémico de meningitis causado por enterovirus, que comprometió a 143 niños de 1 mes a 14 años internados en el Hospital Pediátrico de Posadas (Misiones) con diagnóstico de meningitis aséptica, entre agosto y diciembre de 2005. Se observó un aumento de casos entre las semanas 33 a 50, con un pico máximo entre las semanas 47 y 48, lo que confirmó el brote. La mediana de edad de los niños afectados fue de 8 años y el 55,2% fueron varones. El 80% de los casos se observó entre escolares (5 a 14 años). El promedio del tiempo de internación fue de 4,5 ± 1,7 días, y no se registraron fallecidos. Los LCR se estudiaron mediante examen citoquímico y estudios bacteriológicos y virológicos (aislamiento viral, RT- PCR anidada e identificación molecular mediante secuenciación génica). Los recuentos de células en LCR variaron entre 6 y 5040 células /mm3, el 92% fueron inferiores a 500 células/mm3 y el 43,5% mostró predominio linfocitario. El 56% presentó concentraciones de glucosa normal, con proteínas ligeramente elevadas. El 28% de las muestras estudiadas por cultivo (17/60) mostró efecto citopático, compatible con enterovirus. La RT-PCR anidada permitió detectar enterovirus en un 73% de las muestras (43/59), con 6 casos que se tipificaron como echovirus tipo 4. El índice de positividad al combinar ambas técnicas alcanzó el 83%.

A descriptive retrospective study was carried out to describe an epidemic outbreak of enteroviral meningitis in Misiones. We reviewed records of 143 children from 1 month to 14 years of age who were hospitalized with aseptic meningitis in the Pediatric Hospital of Posadas from August to December 2005. Increased number of cases was observed between weeks 33 to 50 which reached a maximum peak in weeks 47 and 48, confirming an outbreak. The median of age was 8 years old, 55.2% were males. Eighty percent of cases were in 5 to 14 years old children. The average length of time spent in the hospital was 4.5±1.7 days, no deaths were reported. We performed cell counts, chemical and bacterial studies of CSF, and culture or RT-Nested/PCR for enteroviruses. Isolates were serotyped by RT-PCR amplification and genetic sequencing. Cell counts were from 6 to 5040 cells/mm3. Ninety two percent had less than 500 cells/mm3 and 43.5% had lymphocyte predominance. Glucose levels were normal with slightly elevated protein counts in 56% of cases. Of the cultured samples, 28% (17/60) showed cytopathic effect compatible with enterovirus. RT-n-PCR detected enterovirus in 73% (43/59) of the analyzed CSF. Echovirus type 4 was identified in 6 of them. The positive indicator obtained by combining both techniques was 83% (58/70).

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.

An outbreak of echovirus meningitis in children.

An outbreak of aseptic meningitis in children as evidenced by increase in the number of admissions in a tertiary care hospital is described. Clinical data and stool samples were collected from 25 hospitalized infants and young children. The stool samples were subjected to virological investigations. Fever and vomiting were the commonest symptoms. Cerebrospinal fluid (CSF) showed lymphocytic pleocytosis in majority of cases. Of the 25 stool samples, 14 showed an enterovirus specific cytopathogenic effect (CPE) in rhabdomyosarcoma (RD) cell line. All the 14 samples were positive for enterovirus RNA by reverse transcription-polymerase chain reaction (RT-PCR). Partial sequencing of the Virion protein 1 (VPI) region of the enterovirus genome carried out on the first 7 isolates revealed 5 isolates to be echovirus serotype 4 and one each to be echovirus serotypes 3 and 30. All children showed a rapid recovery and were discharged within 3 days of admission.

Echoviroses / Echovirus infections

Trata das echoviroses sob aspectos da etiologia, epidemiologia, patogenia e patologia, manifestaçöes clínicas e tratamento. (MC)

Brote epidémico de meningitis aséptica: experiencias en un hospital infantil privado / Epidemic of aseptic meningitis experience of private hospital

Se informa acerca de las experiencias obtenidas en 193 niños con meningitis aséptica, estudiados durante un brote epidémico registrado entre febrero y julio de 1992. Los cultivos de líquido cefalorraquídeo hechos en el Instituto de Referencia Epidemiológica permitieron identificar al virus Echo 30 como un agente a la meningitis en 48 niños