Enterovirus meningitis in Tunisia (Monastir, Mahdia, 2011–2013): identification of virus variants cocirculating in France

    Enterovirus meningitis in Tunisia (Monastir, Mahdia, 2011–2013): identification of virus variants co-circulating in France Ines Othman, Romain Volle, Aida Elargoubi, Mohamed Neji Guediche, Mohamed Chakroun, Mohamed Tahar Sfar, Bruno Pereira, H´el`ene PeigueLafeuille, Mahjoub Aouni, Christine Archimbaud, Jean-Luc Bailly PII: DOI: Reference:

S0732-8893(15)00390-9 doi: 10.1016/j.diagmicrobio.2015.10.019 DMB 13939

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Diagnostic Microbiology and Infectious Disease

Received date: Revised date: Accepted date:

29 December 2014 10 August 2015 24 October 2015

Please cite this article as: Othman Ines, Volle Romain, Elargoubi Aida, Guediche Mohamed Neji, Chakroun Mohamed, Sfar Mohamed Tahar, Pereira Bruno, PeigueLafeuille H´el`ene, Aouni Mahjoub, Archimbaud Christine, Bailly Jean-Luc, Enterovirus meningitis in Tunisia (Monastir, Mahdia, 2011–2013): identification of virus variants co-circulating in France, Diagnostic Microbiology and Infectious Disease (2015), doi: 10.1016/j.diagmicrobio.2015.10.019

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ACCEPTED MANUSCRIPT Original article Title: Enterovirus meningitis in Tunisia (Monastir, Mahdia, 2011  2013): identification of

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virus variants co-circulating in France

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Running title: EV types and viral loads in CSF specimens Authors

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Ines Othman1,2, Romain Volle3,4,§, Aida Elargoubi5, Mohamed Neji Guediche6, Mohamed Chakroun7, Mohamed Tahar Sfar8, Bruno Pereira9, Hélène Peigue-Lafeuille3,4, Mahjoub

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Aouni1, Christine Archimbaud3,4, Jean-Luc Bailly3 Affiliations

1 Faculty of Pharmacy, LR99-ES27, Monastir, Tunisia

2 Faculty of Sciences of Bizerte, University of Carthage, Tunisia

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3 Université d’Auvergne, EPIE, EA 4843, Clermont-Ferrand, France

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4 CHU Clermont-Ferrand, Service de Virologie, Clermont-Ferrand, France 5 Fattouma Bourguiba University Hospital, Laboratory of microbiology, Monastir, Tunisia

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6 Fattouma Bourguiba University Hospital, Department of Pediatrics, Monastir, Tunisia

Tunisia

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7 Fattouma Bourguiba University Hospital, Department of Infectious Diseases, Monastir,

8 Tahar Sfar University Hospital, Department of Pediatrics, Mahdia, Tunisia 9 CHU Clermont-Ferrand, DRCI, Clermont-Ferrand, France § Current address: Institut Pasteur, Biologie des virus entériques, Paris, France Corresponding author Ines Othman, Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives, Faculté de Pharmacie, Rue Avicenne 5000 Monastir, Tunisie; Phone number: (216) 96539796; Fax number: (216) 73465754; E-mail address: [email protected] Word counts: Abstract, 150; Main text, 2881

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ACCEPTED MANUSCRIPT Abstract Acute enterovirus (EV) meningitis is a frequent cause of hospitalization and a hundred types

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may be involved. 215 patients of all ages with meningitis signs were investigated in two

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Tunisian hospitals. Their cerebrospinal fluid (CSF) was analysed retrospectively for EVs with a TaqMan real-time RT-qPCR. The virus strains were typed and their evolutionary

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relationships were determined by Bayesian phylogenetic methods. An EV genome was detected in 21/215 patients (9.8%). The CSF viral loads ranged between 3.27 and 5.63 log10

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genome copies/mL. The strains were identified in 13/21 patients and assigned to EV-B types. Viruses identified in Tunisian patients were genetically related to variants detected in France. The viral loads were similar in Tunisian and French patients for most EV types. The phylogenetic data and viral loads determined in Tunisian and French patients suggest that

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close EV variants were involved in aseptic meningitis in the two countries over a same period.

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co-circulation

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Keywords: enterovirus meningitis; cerebrospinal fluid; CSF viral load; viral epidemiology;

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ACCEPTED MANUSCRIPT 1. Introduction Enterovirus (EV) infections are frequently involved in diseases of the central nervous

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system (CNS) including aseptic meningitis [1], encephalitis [2], acute flaccid paralysis [35],

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paralytic myelitis [6], and cerebellar ataxia [7]. Aseptic meningitis, the most frequent CNS disease of EV infections occurs as outbreaks of varying size, from local community outbreaks

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to large national epidemics [8,9]. The disease is caused by hundreds of antigenically diverse

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types that are classified into four taxonomic species, EV-A to EV-D [10]. EV sequence typing is required to determine the relationship between virus types and clinical manifestations and to provide evidence-based data for the management of outbreaks. EV infections are reported worldwide [1114] and EV sequence typing and phylogenetic analysis are equally needed to

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identify emerging variants and determine their transmission between neighbouring and distant countries [15].

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In Tunisia, non-poliovirus EV types, notably EV-B, are commonly identified in

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patients with viral meningitis and other neurological disorders [1619]. These types are also the leading cause of aseptic meningitis and a major reason for admission of children and

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adults to hospitals in France [2022]. The aim of this collaborative study was to investigate the epidemiological features of EV meningitis in two Tunisian cities. We tested the CSF of 215 Tunisian patients suspected of having aseptic meningitis for an EV infection and determined the CSF viral load and the type of virus strains in patients with confirmed EV infection. We compared the results with the data of a previous study in French patients [23] and examined virus circulation patterns in France and Tunisia.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Ethics statement

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This study and use of stored samples for testing was approved by the local Tunisian Ethic and

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Research Committee (CHU Fattouma Bourguiba, Monastir; Committee’s advice on 26 June

2.2. Patients and clinical samples

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2014).

The investigation was performed in 215 consecutive patients admitted at the university

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hospital Fattouma Bourguiba of Monastir (n=164) and the hospital of Mahdia (n=51) between January 2011 and January 2013 (Table S1). Patients were eligible for inclusion in the study if there was a suspicion of meningitis based on the clinical judgement of the examining

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physician and as extracted from retrospective medical record review, and a negative CSF bacterial gram stain and culture. The patients (125 male and 90 female) were distributed as

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follows: 10 (5%) neonates (<28 days), 51 (24%) infants (29 days–2 years), 82 (38%) children (2–16 years), and 72 (33%) adults. All patients presented with febrile symptoms and/or signs

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of meningitis, which prompted lumbar puncture by the examining physician either at

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admission or later during the hospital stay. Cytological CSF examination was performed, and protein and glucose concentrations were measured. All the CSF specimens were analysed onsite for biochemical and microbiological testing; the bacteriological cultures were negative. EV PCR testing is not done routinely in the two Tunisian hospitals and thus was performed at the virology laboratory of Clermont-Ferrand, France. The left-over part of CSF was sent in frozen form to preserve integrity to be tested for EV infection, and to determine the viral load and EV type. EV molecular diagnosis was performed in the CSF using the quantitative realtime RT-PCR described below. Complete clinical data were obtained for all patients with positive EV RT-PCR. Data were gathered from medical charts using a standardized questionnaire that included patients’ clinical history, date and time of admission, and CSF

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ACCEPTED MANUSCRIPT parameters. Symptom duration was defined as the time interval between the onset of symptoms and lumbar puncture. Pleocytosis was defined as a CSF white blood cell (WBC)

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count >19 cells/mm3 if the patients were aged ≤28 days, and ≥10 cells/mm3 for the other

neonates <30 days old and ≤0.45g/l for the other patients.

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patients [24]. CSF protein concentration was classified as normal if it was ≤0.9g/l for

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2.3. CSF EV genome testing and viral load determination

Nucleic acids were extracted from 200 µl of CSF by a NucliSens® EasyMAG™ extractor

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(bioMérieux) and eluted in 25 µl of elution buffer. EV genome detection/quantification was done irrespective of cytological examination results with a RT-qPCR assay, developed on the Rotor-Gene 6000 (Qiagen), which is based on uses a hydrolysis TaqMan probe and a competitive RNA internal control [25]. The lowest concentration of viral RNA determined

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with ≥90% probability is 15 copies/μl (95% CI=10–20 copies/μl), i.e. 1875 (3.3

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log10) copies/ml.

2.4. Enterovirus typing by genome sequencing and phylogenetic analysis

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EV strains were typed by sequencing the viral gene encoding the VP1 capsid protein with two

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previously described methods. Enteroviral RNA extracted from CSF specimens was used for partial [26] and complete [27] VP1 gene amplification. The PCR products obtained were subject to nucleotide sequencing on an ABI 3500 DX automated sequencer (Applied Biosystems, Foster city, USA). The nucleotide sequences determined were deposited in GenBank/EMBL/DDBJ under the accession numbers LN713449–60. They were compared with all available nucleotide sequences (as of July 2014) to identify the EV types. The viral sequence in each patient was assigned the type of the GenBank entry that gave the highest identity score [27]. The VP1 sequences determined in Tunisian patients were aligned with those of the homologous EV types determined in our earlier study [23]. The time origin of virus lineages

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ACCEPTED MANUSCRIPT was estimated with a Bayesian phylogenetic method through a Markov Chain Monte Carlo (MCMC) process implemented in the BEAST program v1.8.0 [28,29]. The Bayesian skyline

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model [30] was used as tree prior under a relaxed molecular clock model. MCMC analyses

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were run for 60 million generations, sampling a tree every 3000 steps. Maximum clade credibility (MCC) trees were produced and statistical support for the tree nodes was assessed

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by their posterior probability (pp). Time to the most recent common ancestor (tMRCA) was calculated at relevant nodes of the MCC tree; statistical uncertainty was estimated as a 95%

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highest posterior density (HPD) interval. Unlike the other patients, the viral sequences in patients infected with echovirus type 4 (E-4) were determined partially and as such they were treated separately in the phylogenetic analyses to use the whole genetic information of

2.5. Statistical Analysis

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complete 1D sequences.

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Statistical analysis was performed using Stata software, version 13 (StataCorp, College Station, USA). Quantitative data were presented as numbers and associated frequencies and

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compared in independent groups by the χ2 or Fisher exact test. Quantitative data, were

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presented as the mean ± standard deviation and were compared in groups with the ANOVA or Kruskal-Wallis test. Relations between quantitative parameters were explored by correlation coefficient (i.e. Pearson or Spearman coefficient). The tests were 2-sided and p-value <0.05 was considered as statistically significant.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Patients’ clinical characteristics

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The CSF specimens of 215 Tunisian patients admitted to two hospitals with signs of viral

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meningitis were tested retrospectively for EV with a real-time RT-qPCR assay. CSF tested positive in 21/215 patients (9.8%): 1 neonate, 3 infants, 10 children, and 7 adults (Table 1).

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The neonate and infants are hereafter grouped as infants. Of the 21 patients (female to male ratio=1.33), 9 (42%) were admitted during spring, EV meningitis cases were notified

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sporadically throughout the year but no EV meningitis cases were recorded in October and November. The hospital of Monastir reported more EV confirmed cases than the hospital of Mahdia (table S1). EV-negative specimens did not undergo further analysis to test for other

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viral pathogens. The EV genome was detected below the reproducible detection limit in three other patients, who were therefore excluded from the investigation. The most frequent

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symptoms were fever (n=18/21 patients, 85.7%) and headache (n=16/21, 76.2%). Stiff neck and vomiting were more frequent in children than in adults (Table 1). Symptom duration was

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longer in adults (3.86 days) than in children (2.07 days; p 0.04) (Table S2). The 21 patients

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with laboratory-confirmed EV meningitis spent a cumulative number of 101 days in hospital and they received empirical antibiotics and acyclovir for 38 and 5 days respectively. Six patients including three adults were examined by computed tomography scan of the head, which was normal in all cases. All the patients recovered and no serious complications or sequelae were observed. 3.2. Cytological findings in the CSF of patients with EV meningitis The cytological findings were a pleocytosis (median WBC count, 60 cells/mm3; range, 113800 cells/mm3) in 13/21 patients (62%) and a leukocyte number <10 cells/mm3 in 8/21 patients (38.1%; 2 infants, 2 children, and 4 adults). Symptom duration in the latter group was not different from that of patients with pleocytosis (p 0.52). A 28-day old infant with EV 7

ACCEPTED MANUSCRIPT meningitis had pleocytosis (140 WBC/mm3). None of the patients had low glucose levels in CSF. Protein levels were elevated in 8/21 (38.1%) patients.

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3.3. Virological findings in the CSF of patients with EV meningitis

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Viral strains were typed with the capsid VP1 sequences in 12/21 patients (Table 1). The sequences displayed >87% nucleotide sequence identity with the highest BLAST scoring hits

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(Table S3). Seven EV types were identified, all of which belonged to the EV-B species; echovirus 4 (E-4), the most frequent type, accounted for four cases. Four patients were

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infected with strains assigned to types E-5 (n=2) and coxsackievirus A9 (CV-A9; n=2). Types E-6, E-9, E-18, and E-20 were detected in one patient each. An EV-B strain was revealed in a 13th patient with a sequence amplified from a non-structural viral gene that does not allow identification of EV type (data not shown).

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3.4. Phylogenetic clustering of virus sequences in Tunisian and French patients and

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time origin of virus variants

The phylogenetic comparison of complete VP1 sequences determined in Tunisian and

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French patients [23] is shown in Figure 1. In 2011, two Tunisian patients were infected by a

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same CV-A9 variant (pp=1) which arose in late 2009 (Figure 1A). This virus was genetically related to strains sampled in France at the same period (tMRCA = 2008). An E-18 strain identified in a Tunisian patient in 2012 was genetically distant from a large array of virus variants detected among French patients between 2010 and 2012 (Figure 1B). The E-9 (Figure 1C), E-5 (Figure 1D), and E-6 (Figure 1E) strains detected in Tunisian and French patients displayed a common pattern of close genetic relatedness and recent emergence from a common ancestor. The E-5 strains of Tunisian patients had two distinct ancestors that emerged one to two years before the occurrence of meningitis cases in Tunisia and France. The E-4 sequences were compared with sequences of various geographic origins (Figure 1F). The E-4 strains in Tunisian patients (2012) had a recent common ancestor (pp=1,

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ACCEPTED MANUSCRIPT tMRCA=2009.1) with one of four virus variants detected in French patients since 2008. This heterogeneity reflects the large genetic diversity of E-4 throughout the world over the period

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2008–2012.

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Additional phylogenetic analyses were performed to compare the viral sequences determined in the Tunisian patients with GenBank sequences of various geographic and

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temporal origins (Figure S1). The analyses performed for CV-A9, E-5, E-9, and E-18 showed that the ‘Tunisian isolates’ were always more closely related to the ‘French isolates’ than to

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virus strains sampled elsewhere in the world or at different periods. 3.5. Comparison of viral loads in Tunisian and French patients The viral loads ranged between 3.27 and 5.63 with a mean of 4.27 (median of 4.23) log10 copies/mL of CSF. Mean CSF viral loads differed between patients (Table S2), being higher

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in infants (5.23 log10 copies/mL) than in children and adults (4.02 and 4.08 log10 copies/mL

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respectively, p 0.03). Viral loads did not differ according to whether or not patients had CSF pleocytosis (4.35 vs 4.22 log10 copies/mL). The 8 patients in whom the EV type was not

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identified had lower viral loads than the 13 patients infected with identified EV type (3.75

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[3.275.4] log10 copies/mL vs 4.59 [range, 3.495.63], p 0.01). Three of these eight patients had symptom durations ≥4 days (mean, 5.6 days; range, 47 days) and four had a mean length of hospital stay of 8.3 days (range, 710 days). The viral loads determined for EV types identified in this study were compared with those reported earlier [23]. The viral loads in Tunisian patients were in the previously determined ranges (Table 2) with the exception of patients with E-4 infections (difference in mean viral loads >0.5 log10 copies/mL, p 0.03), which suggests significant variation between the two studies. The untypeable EVs were more frequent in Tunisian (38%) than French (7%) patients. Although the mean viral loads of infections with untypeable EVs were similar in the two studies, the range was wider in Tunisian patients. The CV-A9 infections were related to 9

ACCEPTED MANUSCRIPT lower CSF viral loads in Tunisian than in French patients (4.8 vs 5.68 log10 copies/mL;

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significant variation of –0.88 log10 copies/mL).

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ACCEPTED MANUSCRIPT 4. Discussion The present study shows that EVs were responsible for 9.8 % of cases of meningitis in

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paediatric and adult populations and that sporadic cases of enteroviral meningitis occurred

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throughout the year, as in previous reports [31,18]. A higher prevalence of EV meningitis was observed in the hospital of Monastir than in the hospital of Mahdia. There is no geographical

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explanation for this discrepancy since the two hospitals are in the coastal region of Tunisia. The frequency of patients with laboratory-confirmed meningitis in this study, the age,

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and the admission period were consistent with the features of our earlier study populations in France [23,31] (9.8% vs 8.2%; [28 days–37 years] vs [9 days–57 years]; [January 2011– January 2013] vs [January 2008–December 2012] respectively). In contrast, the time interval

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between the onset of symptoms and lumbar puncture was longer in all Tunisian patients compared with French patients (mean symptom duration 2.66  2 days vs 1.5  1.4 day) and

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accordingly we found no relationship between short symptom duration and the absence of

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pleocytosis. Compared with French patients, those in Tunisia were admitted at a later stage of the disease and symptom duration was longer, two factors which allowed us to show that CSF

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viral load persisted for 6–7 days after the onset of symptoms in two patients. The Tunisian patients also had longer hospital stay. The study was a retrospective analysis and had no impact in patient’s admission. Only patients who required hospitalization were included so the investigation did not evaluate those with mild manifestations and shorter disease durations. However, the study revealed the incidence of EV meningitis in the coastal region of Tunisia. In this region, the incidence of viral meningitis may reflect the transmission of several agents including Toscana virus, West-Nile virus, and EVs [32] as reported for other countries [33]. The patients in this study were not tested for viruses other than EVs but it possible that a number of those with CSF EV loads below the reproducible quantitation limit had another virus as a cause of meningitis. Implementation of rapid diagnosis of viral 11

ACCEPTED MANUSCRIPT pathogens commonly found as a cause of acute meningitis could avert considerable healthcare expenditures, including unnecessary antibiotics and hospital costs.

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Comparison of viral loads determined in this study with those of our previous

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investigation [23] was pivotal to show that the retrospective analysis of CSF was not detrimental for accurate EV genome quantification. A low viral load prevented virus

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identification in eight Tunisian patients. Symptom duration was longer in patients with unidentified EV and so their low viral load may have resulted from clearance of the viral

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infection from the CSF. The virus strains identified in Tunisian patients were assigned to the E-4, E-5, E-9, and E-20 types, which were reported in only 5.3% of French patients [23]. Three types (E-4, E-9, and E20) had already been detected in Tunisia during the period 1992– 2003 [34] and E-9 was regularly detected in the paediatric population of the region of

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Monastir [18,35]. These patterns suggest variations in the frequency of types involved in EV

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meningitis between Tunisia and France.

We provide phylogenetic and virological evidence that a same virus variant of each

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CV-A9, E-5, E-6, and E-9 type co-circulated in the two countries. The virus variants emerged

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only a few years before their spread (tMRCA lower than 3 years before the earliest virus strain within any given phylogenetic group). The viral loads were also strongly suggestive of similarities in EV types detected in patients in the two countries. For instance, the virus in the Tunisian patient with E-6 meningitis was associated with viral loads lower than those of the other types [23]. For E-4, the viral loads were lower in Tunisian than in French patients. This did not result from the presence of PCR inhibitory factors in the CSF and was possibly caused by the high genetic diversity of this type. A similar pattern of high genetic diversity and wide range of viral loads was also determined for E-18. Overall, the phylogenetic and virologic data for types CV-A9, E-5, E-6, and E-9 show co-circulation of virus strains in France and

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ACCEPTED MANUSCRIPT Tunisia and are consistent with possible virus transmission through movement of infected people.

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Further investigations with larger patient populations are now needed to substantiate

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and expand on these findings, including use of similar analyses to determine epidemiologic mechanisms which promote such spread of genetically-related variants. EVs cause a

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significant global burden of infection and some EV variants carry significant morbidity, particularly in vulnerable populations such as children. A combination of clinical, laboratory

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(including phylogenetics), and epidemiological investigations may provide relevant information for monitoring, mapping and even controlling the spread of emerging EV variants

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involved in neurological diseases.

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ACCEPTED MANUSCRIPT Acknowledgements The authors gratefully acknowledge the help of Professor Maha Mastouri (Department of

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Microbiology, Fattouma Bourguiba University Hospital, Monastir, Tunisia) and Pr Hamadi

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Braham (Department of biological analysis laboratory, Tahar Sfar University Hospital, Mahdia, Tunisia) in obtaining patient clinical samples and laboratory data. Ines Othman,

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enrolled in the Faculty of Sciences in Bizerte, University of Carthage, Tunisia received a fellowship grant from the Ministère de l’Enseignement Supérieur et de la Recherche

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Scientifique of Tunisia. This work was supported by grants from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche [grant number EA4843]. The funding sources had no role in study design, the collection, analysis and interpretation of data,

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the writing of the report, and in the decision to submit the article for publication. We thank

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Conflict of Interest

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Jeffrey Watts for help with preparing the English manuscript.

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The authors declare that they have no conflict of interest.

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31. Archimbaud C, Ouchchane L, Mirand A, et al. Improvement of the management of

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infants, children and adults with a molecular diagnosis of enterovirus meningitis

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during two observational study periods. PLoS One. 2013; 8:e68571. 32. Sghaier W, Bahri O, Kedous E, et al. Etude rétrospective des étiologies virales des infections neuroméningées en Tunisie (2003-2009). Med Santé Trop. 2012; 22:373–8. 33. Soares CN, Cabral-Castro MJ, Peralta JM, de Freitas MR, Zalis M, Puccioni-Sohler M. Review of the etiologies of viral meningitis and encephalitis in a dengue endemic region. J Neurol Sci. 2011; 303:75–9. 34. Bahri O, Rezig D, Ben Nejma-Oueslati B, et al. Enteroviruses in Tunisia: virological surveillance over 12 years (1992–2003). J Med Microbiol. 2005; 54:63–9.

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ACCEPTED MANUSCRIPT 35. Gharbi J, Jaïdane H, Ben M’hadheb M, et al. Epidemiological study of non-polio enterovirus neurological infections in children in the region of Monastir, Tunisia. Diag

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Microbiol Infect Dis. 2006; 54:31–6.

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Figure 1 Phylogenies of virus strains assigned to six EV types identified in Tunisian and French patients

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The chronogram trees were inferred with the gene encoding the VP1 capsid protein for coxsackievirus A9 (A), echovirus 18 (B), echovirus 9 (C), echovirus 5 (D),echovirus 6 (E) and echovirus 4 (F). The phylogenetic relationships were inferred with a Bayesian method using a relaxed molecular-clock model. Key nodes with posterior probability (pp) density values > 0.90 are indicated by large circles in purple. The tMRCA values and ranges are indicated for relevant nodes shown with arrows. The arrow heads show distinct virus variants within a type. Each tip branch represents a sampled virus sequence. The branches labelled with green points or coloured in green indicate virus strains identified in Tunisian patients. The sequence names are given as follows: accession number (if this applies)_EV type_virus strain designation_CSF viral load_country with isolation year.

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ACCEPTED MANUSCRIPT Table 1. Clinical data, and cytological and virological findings in the CSF of 21 Tunisian patients. Symptom Viral load Age Sex Symptoms LOS Leukocyte LM PMN Patient duration (log10 a ᵇ c (days) d count (%) f (%) g (days) e copies/mL)

EV type h

1

28 d M

1

4

1

140

65

5.63

E-5

2

37 d F

1,7

8

4

60

90

10

5.40

nd

3

1.2 y F

1,3,4

3

1

0

4

1.7 y F

1,3,4

3

2

9

5

2.8 y F

1,2,4

7

2

21

6

3.2 y M

1,2,3

10

4

7

3.5 y F

1,2,3,4,9

6

2

8

3.6 y F

1,2,4,8

2

2

9

5.7 y F

1,3,4,5

2

0

10

5.9 y F

1,2,3,4,9

3

3

11

7.1 y F

1,2,3,4,5,8 9

1

12

7.5 y F

2,5,6

2

3

13

8.6 y M

1,2,4,5,9

3

14

9.3 y M

1,2,3,5,8

1

nd

nd

5.02

E-5

nd

nd

4.88

E-4

60

40

3.86

nd

3800

40

60

5.05

CVA9

0

nd

nd

3.49

E-6

110

60

40

3.27

nd

74

90

10

4.26

E-4

700

60

40

4.13

CVA9

5

nd

nd

5.42

E-18

35

60

40

3.27

nd

2

30

100 0

3.76

nd

2

21

70

3.73

E-4

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35

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16.6 M 1,2,3,5 8 4 3 nd nd 4.59 EV-B y 16.8 16 F 2,5 3 2 20 80 20 4.56 E-4 y 18.9 17 M 1,2,3,6 10 6 2 nd nd 3.69 nd y 28.0 18 M 1,2,3,4,8 7 4 500 10 90 4.71 E-9 y 32.3 19 F 2,5,9 1 2 0 nd nd 3.51 nd y 35.5 20 M 1,2,5 1 2 0 nd nd 4.23 E-20 y 37.3 21 M 1,2,4,6 8 7 11 100 0 3.27 nd y a Three adults were excluded from the study because the values of CSF EV loads were below the reproducible quantification level of the real-time RTq-PCR assay. M,male; F,female Symptom duration was 5 days (2 patients) and 9 days (one patient). Abbreviations: d, days; y, years.

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ᵇ M, male; F,female ͨ 1,Fever; 2,Headache; 3,Stiff neck; 4,Vomiting; 5,Photophobia; 6,Muscle ache/arthralgia; 7,Vesicular lesion, eruption, rash; 8,Angina/rhinopharyngitis;9,Diarrhea, abdominal pain d LOS, length of hospital stay. e The symptom duration is defined as the time between the onset of symptoms and lumbar puncture. f Percentage of lymphocytes and monocyte cells g Percentage of polymorphonuclear cells. h Abbreviations: nd, not determined; CV-A9, coxsackievirus A9; E, echovirus; EV-B, enterovirus B.

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ACCEPTED MANUSCRIPT Table 2. Comparison of viral loads determined for enterovirus types found in this study with those reported earlier. Viral load, value or range (mean) b Viral Overall viral load load, value or range (mean) b This Earlier This Earlier range (mean) c range study study study study (mean) c 4.10-6.21 4.10-6.21 (4.96)4.102 9 4.13; 5.03 (5.04)4.106.21 (4.96) 6.21 (5.04) 3.73-4.88 5.06-5.49 3.73-5.49 (4.75)3.734 4 (4.33)3.73- (5.19)5.065.49 (4.75) 4.88 (4.33) 5.49 (5.19) 5.11-5.82 5.02-5.82 (5.56)5.022 2 5.02; 5.63 (5.44)5.115.82 (5.56) 5.82 (5.44) 3.3-5.22 3.3-5.22 (3.74)3.3-5.22 1 20 3.49 (3.74)3.3(3.74) 5.22 (3.74) 4.37-5.39 (5.80)4.371 2 4.71 4.37; 5.39 5.39 (5.80) 3.47-5.8 3.47-5.8 (4.61)3.47-5.8 1 11 5.42 (4.55)3.47(4.61) 5.8 (4.55)

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Coxsackievirus A9

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Enterovirus Number of typeEnterovirus type patients

Echovirus 4

Echovirus 6 Echovirus 9 Echovirus 18 Echovirus 20

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Echovirus 5

4.23

na

na

Number of virus variants d variants d 3

4

1

3 2 6 na

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3.3-5.4 3.53-4.33 3.3-5.4 (3.77)3.3-5.4 5 e 5 e (3.7)3.3- (3.86)3.53na (3.77) 5.4 (3.7) 4.33 (3.86) a The viral load values (log10 genome copies per CSF mL) determined in this study were compared with those reported in Volle et al (2014). A difference > 0.5 log10 genome copies/CSF mL between two viral loads was considered significant.> 0.5 log10 genome copies/CSF mL between two viral loads was considered significant. Untypeable enterovirus 8

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b The geometric mean is indicated in parentheses. c The two studies were included to calculate the overall range and geometric mean of viral loads for each enterovirus type. d Number of virus variants among the enterovirus strains related to a viral load in a CSF. A variant was defined as a group of strains that shared a common ancestor, which emerged no more 5 years ealier. e This group also included 6 virus strains related to viral loads below the reproducible detection limit. Abbreviations: na, non applicable.

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Highlights

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 CSF tested positive for EV in 21/215 patients (9.8%).

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 CSF was analysed in 215 Tunisian patients with suspicion of aseptic meningitis.  EV strains in Tunisian patients were closely related to those circulating in France.  CSF Viral loads were similar in Tunisian and French patients for most EV types.

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 Similar EV types co-circulated in France and Tunisia over the period studied.

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