Aseptic meningitis by echovirus 30 in São Paulo state, Brazil

Meningite asseptica por echovírus 30 no estado de São Paulo, Brasil

MEDICAL MICROBIOLOGY

Aseptic meningitis by echovirus 30 in São Paulo state, Brazil

Meningite asseptica por echovírus 30 no estado de São Paulo, Brasil

Bráulio Caetano Machado^*; Raquel de Souza Ferreira; Rita de Cássia Compagnoli Carmona; Maria do Carmo Sampaio Tavares Timenetsky

Instituto Adolfo Lutz, Virologia, Laboratório de Vírus Entéricos, São Paulo, SP, Brasil

ABSTRACT

The objective of this study was to identify and characterize the immunological and molecular aspects of Human Enterovirus (HEV) associated with aseptic meningitis cases in São Paulo State, Brazil. Clinical samples (cerebrospinal fluid CSF, stool and sera) were analyzed from 21 viral meningitis suspected cases collected in the period from 1998 to 1999. The isolated virus were identified as belonging to Enterovirus genus and specifically to echovirus serotype 30 (EV-30), using immunological techniques as Indirect Immunofluorescense Assay (IFA) and Neutralization (Nt) and by molecular technique of Reverse Transcription Polymerase Chain Reaction (RT-PCR) directed to the 5' NTR and VP1 regions of viral genome. The results obtained contributed with to the surveillance system of São Paulo State and they are important due to a shortage of epidemiological data about the circulation of EV-30 associated to aseptic meningitis in this region of the world.

Key words: Enterovirus, viral meningitis, molecular and immunological diagnosis, RT-PCR for Enterovirus, indirect imunnofluorescense assay

RESUMO

O objetivo deste estudo foi identificar e caracterizar quanto aos aspectos imunológicos e moleculares os Enterovirus humanos associados a casos de meningite asséptica. Amostras clínicas (líquido cefaloraquidiano, fezes e soro) originárias de 21 casos suspeitos de meningite viral, provenientes do estado de São Paulo no período de 1998 a 1999 foram analisadas. Os vírus isolados foram identificados como pertencentes ao gênero Enterovirus e mais especificamente ao sorotipo echovírus 30 (EV-30), utilizando-se técnicas imunológicas como Imunofluorescência Indireta e Neutralização e técnica molecular da transcrição reversa - reação em cadeia da polimerase (RT-PCR) direcionada para as regiões 5' não traduzida (5'NTR) e Proteína Viral 1(VP1) do genoma viral. Os resultados obtidos contribuíram com o sistema de vigilância epidemiológica do estado de São Paulo na geração de informações sobre a circulação de EV-30 associado à meningite asséptica nessa região do mundo.

Palavras-chave:Enterovirus, meningite viral, diagnóstico imunológico e molecular, RT-PCR para Enterovirus, imunofluorescência indireta

INTRODUCTION

Aseptic meningitis is a central nervous system infection occurring sporadically, and as outbreaks, presenting more than 90% of cases with an identified cause associated with enteroviruses (31). Human Enterovirus (HEV) is a RNA positive single stranded virus in the Picornaviridae family, constituted of five species grouped into (HEV-A, -B, -C, -D and Poliovirus) clusters. The HEV B cluster includes coxsakievirus B (CV-B), coxsackievirus A9 (CV-A9), enterovirus 69 (HEV-69), and all echoviruses (EV) (29).

Although most of HEV infections cases are asymptomatic or result in mild febrile illnesses, aseptic meningitis is predominantly reported. The HEV strains most frequently isolated from patients with aseptic meningitis are EV-30, EV-6, EV-11, and EV-9 (6).

Analyzing notified meningitis cases in the period from 1979 to 1999 in São Paulo State, Brazil, we observed a high incidence of meningitis cases without determined etiology, and among them, 50% to 60% of probable viral etiology (10).

In 1998 an increase of aseptic meningitis cases was observed in the city of São Paulo with a total of 101 notified cases until October denoting an outbreak in progress.

Laboratory data (CSF cerebrospinal fluid, cytochemical diagnosis) and epidemiological surveillance of the cases indicated viral association and other arising of another outbreaks were further observed in several cities of São Paulo State. (CVE, unpublished data, 1998). These data demonstrated the need for research of viral agents associated to non-bacterium meningitis in our region.

With the objective to investigate the etiological viral agent of new suspected cases and to perform this characterization, we received samples of 21 patients from different affected regions of the State. The evaluation of laboratory results obtained was made with epidemiological relationship of the cases and the clinical diagnosis from medical services of these regions.

Molecular methods for HEV detection are increasingly becoming available (30, 31). Serotype-specific PCR (Polymerase Chain Reaction) primers have been developed by CDC (Centers for Disease Control and Prevention, USA) for several enteroviruses, including EV-30 (15). These serotype-specific primers are useful for rapid differentiation of outbreak strains from sporadic infections with other enteroviruses.

In the present study we describe the immunological and molecular characterization of HEV isolates obtained from viral meningitis suspected cases collected in the period of the outbreak, from November 1998 to February 1999.

MATERIALS AND METHODS

Clinical Samples

Case definition of aseptic meningitis was based on clinical and cerebrospinal fluid (CSF) findings compatible with viral meningitis and tested negative for other pathogens.

Stool, and CSF samples from 21 patients with clinically suspected aseptic meningitis were sent for viral diagnostic. Sera samples from acute and convalescent phase were also collected in 11 cases to perform antibody seradiagnosis.

Viruses

Virus prototype strains (EV-30 and EV-4) were obtained from the American Type Culture Collection - ATCC (Rockville, Md.) and HEV isolates from clinical samples maintained in our laboratory.

Viral culture

The samples were collected in sterile containers, and transported to laboratory adequately at -195ºC (liquid nitrogen) for CSF and 0ºC to 4ºC for stool and sera in expanded polystyrene boxes. CSF was cultured without treatment, and stool samples were prepared with 10% suspension in phosphate-buffered saline (PBS) following 60 minutes at 10,000 X g centrifugation, and the addition of 0.1 mg of antibiotic solution (penicillin and streptomycin, at final concentration of 50 µg per mL), to the supernatant. The inoculum was stored at -70ºC and inoculated in cell culture. Viruses were propagated in Vero (green monkey kidney) ATCC-CCL-81, RD (human rabdomyosarcoma) ATCC-CCL-136, and HEp-2 (human larynx carcinoma) ATCC-CCL-23 cell monolayer cultures. These cultures were inoculated with a minimum of 0.1 mL of specimen and after 30 minutes of adsorption, were maintained in Eagle L15 medium incubated at 37ºC in bacteriologic incubator. The cells were observed daily for the presence of viral cytopathogenic effect (CPE) (8,21).

Indirect Immunofluorescence Assay

Positive cell cultures were harvested when 75% of cytopathogenic effect was attained. Cell supernatant was centrifuged at 3,000 X g for 15 minutes, then discharged and the cell pellet was seeded onto slides, dried, and fixed in cold acetone at 4ºC. Monoclonal antibodies (Mabs) (Chemicon Inc., Temucula, CA) directed to Enterovirus (genus, group and serotypes) and a goat anti-mouse IgG antibody conjugated with fluorescein isothiocyanate were used according to the manufacturer's instructions. Incubations were performed at 37ºC for 30 minutes in a moist chamber. After being washed in PBS, the slides were mounted with buffered glycerin and observed under UV light with an epi-fluorescence microscope at a magnification of X 400 (3).

Neutralization test (Nt)

Serotyping was carried out using a neutralization method with the use of intersecting pools of antisera A to H (Statens Serum Institut, Copenhagen, Denmark) against the titulated virus, 100TCD₅₀, by Reed Muench method (28) according to the Lim-Benyesh-Melnick (LBM) scheme for identification (19). Individual antisera against coxsackievirus B3 (CV-B3) was also included.

Primers

PCR primers were selected from highly conserved regions of the 5'NTR (nontranslated region): ENV-AS1 anti-sense (5'-ATTGTCACCATAAGCAGCCA-3') and ENV-S1 sense (5'-CAAGCACTTCTGTTTCCCCGC-3') for characterization of Enterovirus genus (ROTBART, H. A. 1990). To characterize echovirus serotype 30, primers directed to VP1, EV-30 P1 (5'-TCCGCGTGCAACGATTTCTC-3') and EV-30 P2 (5'-CTCCCACACGCAGTTCTGCC-3') were selected respectively (2).

RNA extraction and in house RT-PCR

Viral RNA was extracted from infected cell culture supernatant with Trizol LS (Invitrogen) according to the manufacturer's instructions. Reverse transcription-PCR (RT-PCR) was carried out by a single-step method. In the assay the reverse transcription and molecular amplification steps are combined through as described previously with some modifications (2). To 5 µL of the RNA was added 45 µL of the RT-PCR mixture containing 10x PCR buffer (TRIS-HCl 100 mM, KCl 500 mM, Triton X-100 1%), MgCl₂ (25 mM), mixture of dNTP (1.25 mM each dATP, dGTP, dCTP, dTTP), pool of primers (AS1 - S1 or EV-30 P1 EV-30 P2, 20 µM/µL each); Taq DNA polymerase (2.5 U/µL), Super Script II Reverse Transcriptase (1U/µL, Invitrogen Life Technologies), RNAsin (40,000 U/mL, Pharmacia), and H₂O. The mixture was incubated in thermocycler (Perkin Elmer 9600) for 60 minutes at 42ºC, and followed by 25 cycles (1 minute at 94ºC, 2 minutes at 50ºC, and 1 minute at 72ºC). Following this process, 5 µL of the PCR products were subjected to electrophoresis in 1.5% agarose gels (Ultra Pure, Invitrogen) in TBE buffer (Tris, boric acid, EDTA) containing Ethidium Bromide (0.5 µg per mL), and was run for 50 minutes at 150V. PCR products were photographed under UV light. Amplicons of 437 bp and 900 bp were amplified from each viral cDNA, using primers for 5'NTR and for VP1 regions, respectively (30).

Antibody titration in paired sera

For serological diagnosis, paired sera samples were required. This microneutralization technique was applied for detection of antibodies against the prototype antigen (EV-30). To perform the test, serial dilutions from 1:8 to 1:1024 (two fold) of the paired sera, were mixed to the antigen (100TCD50/0.1 mL) and incubated for 2 hours at 37ºC. After the incubation period, 100 µL of RD cell suspension (220,000 cells per mL) was distributed into the wells. Microplates were incubated for 72 hours (37ºC) and observed on the inverted microscope for viral infectivity neutralization. The increase of 4x in the antibody titer was considered indicator of seraconversion (21).

Ethical Aspects

This study was submitted to the Ethical Research Committee of the Adolfo Lutz Institute and was approved.

RESULTS

Twenty-one suspect viral meningitis cases from several regions of São Paulo State were studied. Eighteen CSF samples, 14 stools samples and 11 paired sera samples were analyzed. The results obtained are presented in Table 1.

The isolated viruses were identified as Enterovirus, echovirus serotype 30, in 62% (13/21) of the studied cases.

Viral isolates were observed after 2 passages in RD cell line. Virus culture from CSF was obtained after the first or second passage in cell culture; virus culture from stool needed one more passage due to anti-cell toxicity, even with treatment of stool material. HEV were isolated in 44% (8/18) of CSF samples and in 57% (8/14) of stool samples. No other virus was isolated from the different cell lineages used.

Of the total 11 cases with paired sera samples, 10(91%) presented significant conversion in the antibody titer for the respective prototype strain (EV-30) and 1 case presented the same antibody titer in both samples collected in the acute and convalescence phases. Subsequently, these paired sample presented considerable seraconversion for the isolated virus (EV-30) originated from another case of the outbreak.

By IFA technique, 16 viral isolates were identified as Enterovirus genus, echovirus group and EV-30 serotype. Two isolates presented cross-reaction to specific monoclonal antibody for EV-4 serotype. These isolated viruses were neutralized by CEG pools of (LBM) specific sera for HEV and were identified as EV-30 by Neutralization (Nt).

Using PCR test, all isolates were genotyped as Enterovirus genus, presenting cDNA amplicons of 437bp for the region 5'NTC, and as EV-30 serotype presenting amplicons of 900bp for the VP1 region. The specific primers for the VP1 region used in this study did not amplify the EV-4 prototype virus. Figs. 1 and 2.

DISCUSSION

In this study we describe the identification and characterization of HEV isolates during an outbreak of aseptic meningitis in São Paulo State, Brazil.

The isolated viruses during in this period were identified as Enterovirus genus, echovirus group and EV-30 serotype using, both, molecular and immunological techniques.

Analyzing our laboratory results associated with epidemiological data and clinical diagnosis information of each case, we conclude that Enterovirus EV-30 was the etiological agent involved in the outbreaks in the State of São Paulo.

This study points out a period of increase of viral meningitis cases in the State of São Paulo according to epidemiological studies of CVE. For this period (1998 and 1999) the incidence was 17,00/100.000 [cases per inhabitants] and the incidence media for viral meningitis for the last 15 years (1990 to 2005) was 11,74/100.000 (27).

The result signs the importance of echovirus serotype 30 association in aseptic meningitis cases adding new data collected in this region of the world, which is characterized by rather poor information about HEV circulation. In Brazil we have few articles related to EV-30 meningitis (16,32). The circulation of echovirus, specifically echovirus 30 is fully worldwide related nowadays.

During the period from 1970 to 2001, in the USA, EV-30 was among the 15 enteroviruses most commonly reported each year, accounting for 8.2% of all enteroviruses isolates reported to CDC (CDC, unpublished data, 2003). The increase of aseptic meningitis associated with high activity of EV-9 and EV-30 is consistent with the historical data; during 1998 and 1999, the peak years for viral meningitis hospitalizations in the United States coincided with periods of high activity of either EV-9 or EV-30 (6). In Spain a study reported cases associated to viral meningitis, during the period from 1988 to 1997 with a high incidence of HEV and the predominance of EV-30 (90%) among non-polio serotype (36). This serotype was responsible for aseptic meningitis epidemics in Japan in 1991 and 1997 (39, 40), Switzerland in 1996 (13), Canada in 1998 (4), Romania in 1999 (5), and more recently published, in Turkey in 1999 (26), France in 2000 (7), Belgium in 2000 (35) and Brazil in 2003 (16,2). Our retrospective study agrees with literature data.

Isolation of HEV from affected organs and from body fluids (e.g.:CSF), gives the strongest evidence for HEV infection (9,22). However, stool sample is the most sensitive for HEV detection in cell culture due to more amount of viral particles excretion in a 30 day period (38).

Although viral isolation in cell culture is often being used as a gold standard for HEV identification, the technical procedures and information about viral isolation could vary considerably between laboratories (11,18).

In this study, three types of cell lineages were used. They were susceptible to isolation of the most viruses associated to neurological disease. All echovirus serotype 30 were isolated in RD cell culture, according to literature data (14).

The etiology of meningitis cases was determined by detection of virus in CSF in stool and antibody titer conversion for isolated virus in cases which we had stool material isolation only.

Identification of HEV in CSF defines the agent, but, on the other hand, the isolation of virus in stool needs antibody titration of the patient's paired sera to confirm the viral etiology. This study demonstrated the importance of seraconversion and virus isolation from stools samples to confirm the etiology in five cases with stool positive cultures. Sub clinical infections are very common due to a frequent exposure to HEV and the results could be confused.

The introduction of IFA allowed serotyping of isolated virus with reducing of time and brought agility to the release of results; but, it is important to note that, IFA can present, in some cases, false negative results. Some viral strains can present low affinity to a specific monoclonal antibody due to mutations leading to antigenic changes in the target epitope occurring, therefore, not recognition by monoclonal antibody (3). Specific MAb directed to EV-4 can present low cross-reaction with EV-30, EV-6 and EV-11 MAbs (3,16). In our study cross-reaction between EV-4 and EV-30 MAbs was observed in 2 samples. Serotypes were defined in complementary tests, Nt and PCR.

The virus was identified as echovirus serotype 30 by Neutralization (Nt) (17), the gold standard method to HEV identification. Antigenic changes among HEV serotypes can cause difficulties in identification by traditional Nt test (37,38).

Although serotyping may in general be of limited importance for the management of patients with an HEV infection, it is essential to understand the epidemiology of HEV for monitoring the emergence of new serotypes or possible changes in virulence, allowing previous and fast detection of new variants (12,34).

The choice of PCR testing has been increasing for HEV infection diagnosis, particularly for patients with aseptic meningitis. It has been demonstrated to be more sensitive than viral isolation, with 30 to 100% more of positive results (1,30,33,41).

On the other hand, the use of cell culture promotes viral replication and increase of viral mass allowing the serotyping process through IFA and Nt.

In our study the use of PCR test for the 5' NTR region demonstrated to be of great utility, for viral identification after the first passage in cell culture increasing the sensitivity of viral detection and decreasing the time of detection. The 5'NTR region sequence presents high grade of homology for almost all HEV and therefore, PCR amplification of this region was carried out to identify Enterovirus genus.

Early etiologic diagnosis of aseptic meningitis helps to avoid unnecessary antibiotic treatment and additional testing.

Correlation between VP1 and serotype has been demonstrated by comparison of VP1 gene sequences of all HEV prototypes (24). Sequences of the VP1 region from isolated virus have been analyzed to genomic comparison with HEV detected worldwide. Serotype-specific PCR developed for molecular typing of EV-30 isolates had become a rapid molecular biology-based typing method available (23). Adoption of this technique for identification allowed to clarify cases of cross-reaction observed between EV-30 and EV-4 and the reduction of time for laboratory diagnosis.

Molecular diagnosis directed to VP1 target gives typing results corresponding to serotype determined by IFA and Nt with type-specific antisera (24). Sequencing isolates with generic primers that amplify all HEV serotypes has been established as a new and useful tool for molecular typing (25). These new primers successfully amplified HEV isolates of all 64 serotypes. When compared with Nt or IFA this test provides more advantages for a wide-ranging serotype investigation.

Molecular results confirm the EV-30 as etiological agent of the cases concluding our studies and contributing with epidemiological data about EV circulation in viral meningitis outbreaks at the region of São Paulo State, Brazil.

ACKNOWLEDGMENTS

We would like to thank the staff of the Cellular Cultures Section of Adolfo Lutz Institute for providing the cells used in this study. Rubia Anita Ferraz Santana and Denise Hage Russo for the intense collaboration and motivation on this work.

Submitted: April 20, 2006; Returned to authors for corrections: June 29, 2006; Approved: October 13, 2006

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Publication Dates

• Publication in this collection
  24 Apr 2007
• Date of issue
  Mar 2007

History

• Accepted
  13 Oct 2006
• Reviewed
  29 June 2006
• Received
  20 Apr 2006