Clinical and virological features of an aseptic meningitis outbreak in North-Eastern France, 2005

Journal of Clinical Virology 42 (2008) 225–228

Short communication

Clinical and virological features of an aseptic meningitis outbreak in North-Eastern France, 2005 Delphine Brunel a,c , Nicolas L´evˆeque a,b , J´erˆome Jacques a,b , Fanny Renois a,b , Jacques Motte c,d , Laurent Andr´eoletti a,b,∗ a

c

Laboratoire de virologie m´edicale et mol´eculaire, Centre Hospitalier Universitaire de Reims, France b IFR 53/EA-3798 (DAT/PPCIDH), Facult´ e de M´edecine de Reims, France Service de P´ediatrie A, American Memorial Hospital, Centre Hospitalier Universitaire de Reims, France d INSERM Unit´ e 666 Strasbourg, France Received 5 February 2008; accepted 8 February 2008

Abstract Background: Enteroviruses (EVs) are considered as a major viral etiological cause of aseptic meningitis in children. Objectives: We assessed the clinical and virological features of an aseptic meningitis outbreak in North-East of France, 2005. Study design: Classical bacteriological analysis, Herpesviridae and EV PCR assays had been prospectively performed on cerebrospinal fluid (CSF) samples taken from 80 children hospitalized for aseptic meningitis. For each EV strain identified as etiological agent, a phylogenetic comparison of partial EV VP1 capsid protein coding gene was performed. Results: The children older than 12 months (n = 75) presented a typical aseptic meningitis syndrome, whereas the children aged less than 1 year (n = 5) demonstrated only fever and hypotonia. Among the 80 studied children, EV was identified as the etiological cause of aseptic meningitis in 73 (91%) cases. Echovirus 30 (E30) was the most common isolated serotype (84% of 51 EV strains). VP1 phylogenetic analysis revealed that E30 strains were genetically closer to those isolated during 2000 aseptic meningitis outbreak comparatively to those identified during 2003 and 2006 non-epidemic years. Moreover, the genetic study demonstrated the co-circulation of four distinct lineages without any difference in temporal distribution or clinical features during the 2005 outbreak. Conclusions: The present report demonstrates the co-circulation of distinct E30 lineages during the same aseptic meningitis outbreak season. This E30 genetic diversity may be a prerequisite for the emergence of new strains potentially responsible for further aseptic meningitis outbreaks. © 2008 Elsevier B.V. All rights reserved. Keywords: Enterovirus; Aseptic meningitis; Phylogenetic analysis; Echovirus 30

1. Introduction Aseptic meningitis is commonly defined as a syndrome characterized by acute onset of signs and symptoms of meningeal inflammation, cerebrospinal fluid (CSF) pleocytosis and the absence of microorganism on Gram stain and/or routine culture (Cherry, 2004). Aseptic meningitis is frequently caused by viral agents, particularly human ∗ Corresponding author at: Laboratoire de virologie m´ edicale et mol´eculaire, Service de Microbiologie, Hˆopital Robert Debr´e, Avenue du G´en´eral Koenig, 51092 Reims Cedex, France. Tel.: +33 3 26 78 39 93; fax: +33 3 26 78 41 34. E-mail address: [email protected] (L. Andr´eoletti).

1386-6532/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2008.02.008

enteroviruses (EVs) (King et al., 2007; Michos et al., 2007). Transmitted through the fecal oral route and potentially by respiratory route, EVs are the pathogen associated most commonly with acute meningitis worldwide and can cause sporadic cases, outbreaks and epidemics (Antona et al., 2007; Tseng et al., 2007). These epidemics typically occur during summer and fall in pediatric patients (Antona et al., 2007). Since the development of RT-PCR assays for their detection and their typing from the clinical samples, many aseptic meningitis outbreaks were described worldwide during the last few years (Mirand et al., 2007; Zhao et al., 2005). However, the virological mechanisms responsible for the emergence of EV neutrotropic epidemic strains remains poorly understood. During the 2005 summer season,

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enteroviruses were responsible for a major outbreak of acute aseptic meningitis in French children (Antona and Chomel, 2005; Mirand et al., 2006). The present study describes the clinical and virological features of this aseptic meningitis outbreak in North-East of France.

2. Patients and methods 2.1. Patients The study retrospectively identified and reviewed 80 children ≤18 years of age admitted from April to November 2005 with a diagnosis of aseptic meningitis to the paediatric emergency unit of four hospitals in the North-East region of France (Reims, Charleville-M´ezi`eres, Troyes and Soissons, France). Aseptic meningitis was defined as an acute illness consisting in symptoms and signs of meningeal irritation, headache, nausea, vomiting and fever associated to a white blood cells count (WBCC) in the CSF of ≥10/mm3 . Demographic, laboratory and clinical data were then collected. For each child, inform consent was obtained from their family or their relatives. The present study was performed in the university medical hospital of Reims (Champagne-Ardenne, France) and was approved by the hospital’s ethics committee. 2.2. Microbiological analysis of paediatric samples All children underwent a CSF puncture which was routinely tested for bacteria while enterovirus (Enterovirus Consensus kit, Argene Biosoft, Varhiles, France) and Herpesviridae (Herpes Consensus Generic, Argene Biosoft, Varhiles, France) PCR tests were performed according to the manufacturer’s instructions. Throat and faeces specimens, obtained from 55 and 31 children, respectively, were cultured for virus isolation as previously described (Freymuth et al., 1997). In case of isolation, enteroviruses were typed either by the conventional neutralisation assay after isolation or by direct sequencing of the 3 end of the VP1 capsid coding gene as previously described (Melnick et al., 1977; Oberste et al., 2003). 2.3. Phylogenetic comparison of partial enterovirus VP1 capsid protein region Total RNA was extracted from 140 ␮l of CSF or cell culture supernatant of throat or faeces samples using the QIAamp viral RNA minikit (Qiagen, Courtaboeuf, France). RT-PCR amplification and sequencing of partial VP1 capsid coding gene were carried out as described previously (Oberste et al., 2003). The sequences were manually corrected and compared for identification by sequential pairwise alignment with all the enterovirus sequences available in GenBank using BLAST. Twenty-two partial VP1 sequences were then aligned using the computer

program Mega, Version 4 (S. Kumar, K. Tamura, I. Jakobsen, M. Nei; available at http://www.megasoftware.net) with 24 E30 strains isolated in North-Eastern France from 1999 to 2006. The phylogenetic tree was built using the MEGA 4 program using the neighbour-joining method as implemented in the Mega computer program (Saitou and Nei, 1987). VP1 sequences obtained from the study are available in the EMBL database under the following accession numbers: AM492322–AM492331, AM492333–AM492335, AM492337–AM492343, AM492345–AM492353, AM492357–AM492358, AM492360, AM492363–AM492364, AM492367–AM492368, AM492371–AM492377, AM492380, AM937003–AM937005.

3. Results 3.1. Clinical and virological findings The majority of the patients older than 12 months (n = 75) presented a typical syndrome characterized by acute onset of headache (100%), fever (89.7%) and vomiting (85.3%). In children less than 1 year (n = 5), fever and hypotonia were the only reported symptoms whereas signs of meningeal infection were missing. Only one fatal case of EV-related leukoencephalitis was developed in 18 months infant during the present outbreak (Brunel et al., 2007). EVs were identified as etiological cause of aseptic meningitis in 73 of 80 (91%) cases, due to either a positive RT-PCR test (n = 70) or a positive culture isolation from a throat or stool specimens (n = 3 cases). HHV6 (n = 2) and VZV (n = 1) were identified in 3 (4%) children including a mixed infection with HHV6 and EV strains in a CSF sample. Of the 73 EVs related aseptic meningitis, 51 strains were identified. Echovirus 30 (E30) was the most common isolated serotype (84%) and was thus considered as responsible for the outbreak, while Echovirus 18, 13, 6, 3 and Coxsackievirus A16 only caused sporadic cases (6%, 4%, 2%, 2% and 2%, respectively). Only 5 (6%) of 80 aseptic meningitis cases remained without any etiological characterization. 3.2. Phylogenetic comparison of enterovirus partial VP1 capsid protein region Partial VP1 sequences were determined for 22 E30 strains isolated during the 2005 outbreak and compared with 24 E30 strains isolated from 1999 to 2006. Phylogenetic analysis revealed that 2005 strains were genetically closer to those isolated from patients involved in 2000 outbreak than to 2003 and 2006 non-epidemic strains. Interestingly, our VP1 genetic analysis demonstrated the co-circulation of four distinct lineages (bootstraps > 70%) during the 2005 outbreak (Fig. 1). These lineages did not display any differences in their temporal distribution, biological or clinical features (not shown). Comparative analyses with E30 strains isolated from

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Fig. 1. Phylogenetic tree depicting the relationships between partial VP1 capsid gene sequences of 46 French E30 strains isolated from 1999 to 2006. Only bootstrap values ≥ 70% are indicated. Each strain is referenced by its EMBL accession number. For E30 strains isolated in 2005, the month of isolation is notified. The four different lineages detected during the 2005 outbreak are depicted by different symbols: (䊉), (), (), ().

1999 to 2006 of amino acid changes within the 3 end of the VP1 revealed that all the E30 strains detected in 2005 were characterized by one amino acid substitution (N89S) associated to second one (F118L) only in the fourth lineage (Fig. 1). Interestingly, these two amino acids have been shown as belonging to a part of the poliovirus neutralizing antigenic site I (Minor et al., 1986).

4. Discussion Since the surveillance of enteroviruses in humans was reinforced in France in 2000, E30 was the main non-polio enterovirus identified (Antona et al., 2007). E30 is known to cause outbreak through maintaining their isolation rates and increase their circulation periodically (Chomel et al., 2003; Zhao et al., 2005). In the present work, we assessed the clinical and virological features of the outbreak of aseptic

meningitis due to E30 that occurred during summer 2005 in North-Eastern France. Interestingly, this study revealed phylogenetic relationship between 2005 and 2000 strains, two epidemic years, whereas we observed a lack of clustering with strains from previous and following non-epidemic years (Fig. 1), suggesting the existence of specific epidemiological properties of some strains to cause epidemics. A typical aseptic meningitis syndrome was diagnosed in 75% of the study cases, whereas only one fatal leukoencephalitis was developed in 18 months infants during this 2005 French outbreak (Brunel et al., 2007). In the present report, we evidenced the co-circulation of four distinct lineages within the same local geographical area during the 2005 outbreak that could be directly related to the genetic drift of EV genomic RNA (Rico-Hesse et al., 1987; Takeda et al., 1994). This multiplicity of E30 genetic variants during a same epidemiologic season has been previously characterized during local meningitis outbreaks (Mirand et al., 2006; Savolainen et al., 2001). This phenomenon could

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impact on the mechanisms of neutralizing antibodies recognition or on the virus–target cell interactions. Interestingly, E30 variants detected in Champagne-Ardenne presented several nucleotide and two amino acid changes within the 3 end of the VP1 in comparison to previous circulating strains. Because amino changes were localized within capsid highly antigenic site, these viruses could have improved their fitness and increased their pathogenicity by immunological escape mechanisms (Minor et al., 1986). In order to explore this hypothesis, larger antigenic variations should be assessed through the complete capsid protein coding gene sequencing in further investigations. Moreover, because E30 has been shown to recombine extensively with other HEV-B strains in the non-structural region, the observed co-circulation of at least six different EV serotypes made possible recombination events within a same infected host during a same epidemic season (Lindberg et al., 2003; Lukashev et al., 2003, 2007). In order to determine the existence of such recombination events, further analysis of the 3D genome region would be useful among the study strains of each epidemic and nonepidemic year. In conclusion, the present report demonstrates the cocirculation of distinct E30 lineages during the same epidemic season. This E30 genetic diversity may be a prerequisite for the emergence of new strains responsible for further potential aseptic meningitis outbreaks. The present study highlights the need for a sentinel laboratory network for a national surveillance of clinical and epidemiological features of EV neurological paediatric infections.

Conflict of interest None of the authors of the present manuscript have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy).

Acknowledgments We are indebted to the paediatricians who had included the patients in to the present study. This work was supported in part by a grant for clinical and virological research (EA-3798: DAT/PPCIDH) from the Medical University and School of Medicine of Reims, France.

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