Epidemiological, Molecular, and Clinical Features of Enterovirus Respiratory Infections in French Children between 1999 and 2005

Study population.From January 1999 through December 2005, 11,509 consecutive children (mean age, 2.3 years; age range, 9 days to 15 years) attending one of the five referent pediatric emergency departments of the region Champagne Ardenne (France) were prospectively enrolled in a north French Caucasian pediatric cohort for the exploration of viral respiratory diseases. For each child, informed consent was obtained from their family or relatives. The present study was conducted by the university medical hospital of Reims (Champagne Ardenne, France) and was approved by the hospital's ethics committee. All children underwent general, neurological, and respiratory examinations by a pediatrician, who carried out a nasopharyngeal aspirate sampling for the detection of common respiratory human viral pathogens by classical cell culture and immunofluorescence assays (17, 19). Moreover, according to the clinical symptoms observed, cerebrospinal fluid, peripheral blood, throat, or urine samples were also concomitantly sampled and tested by standard culture assays for the detection of classical human bacterial or viral pathogens, by standardized reverse transcription-PCR (RT-PCR) assays for the detection of EV strains in cases of aseptic meningitis or by standardized PCR assays for the detection of Mycoplasma sp. or Chlamydia pneumoniae in respiratory tract samples (9, 18). Among these 11,509 consecutive children enrolled in the study cohort, 252 (median age, 5.1 years; age range, 14 days to 15 years; male/female ratio, 159/93) were retrospectively selected because (i) they were aged ≤15 years; (ii) EV was the unique infectious agent detected in their nasopharyngeal samples as determined by classical cell culture assays and then identified as the etiological cause of the clinical syndrome diagnosed at the time of emergency visit; and (iii) they were free of cystic fibrosis, bronchopulmonary dysplasia, congenital heart disease, and systemic glucocorticoid treatment or with a chronic genetic or acquired immunodepression.

During the present study, 61 children were excluded because they demonstrated mixed respiratory infections (n = 33) or because they were suffering from chronic genetic or acquired respiratory pathologies or immunological immunodepression (n = 28) (data not shown). Table 1 presents the clinical features based on discharge diagnosis for the selected population.

Nasopharyngeal sample collection.Nasopharyngeal secretions were collected from all of the studied patients using 2 ml of sterile physiological saline fluid with a disposable mucus extractor at time of hospital admission (2). Nasopharyngeal wash fluids were then diluted in 3 ml of virus transport medium (0.5% bovine serum albumin, 1.500 U of penicillin, 1 ng of streptomycin, minimal essential medium, and 4.76 mg of HEPES in 2 ml of tryptose phosphate broth) and divided into aliquots into two separate sterile tubes. One tube was directly used to perform immunofluorescence detection of viral antigens and cell culture detection assays; the second tube was immediately frozen and stored at −80°c prior to molecular assays (17, 20).

Classical cell culture assays for virus isolation.For each studied patient, 200 μl of nasopharyngeal secretion sample diluted in viral transport medium was inoculated in duplicate onto 24-well plates covered with monolayers of continuous human diploid fibroblasts (MRC-5), rhesus monkey kidney (MA-104) and Madin-Darby cell kidney (MDCK) cells as previously described (17). Each plate was incubated for 8 days at 37°C in a 5% CO₂:95% air atmosphere. The plates were examined daily under light microscopy to detect the presence of a cytopathic effect on the cell culture monolayers. Two subcultures were performed at days 8 and 16 for each well as described above. EV isolates were typed by the standard method neutralization with EV type-specific antisera (17, 19, 26).

RNA isolation.Total RNA was extracted from 140 μl of culture supernatants by using the QIAamp viral RNA minikit (Qiagen, Courtaboeuf, France), according to the manufacturer's recommendations. Nucleic acids were eluted in a final volume of 50 μl of diethyl pyrocarbonate-sterile water, as described by the manufacturer's recommendations. The nucleic acid concentration was estimated by spectrophotometric measurement (optical density) at 260 nm before the samples were divided into aliquots and stored at −80°C until used (17).

Phylogenetic comparison of partial EV VP1 capsid protein region.RT-PCR amplification and sequencing of a part of the VP1 capsid gene were carried out as described previously (30). The sequences were manually corrected and aligned by using the computer program MEGA 3.1 (S. Kumar, K. Tamura, I Jakobsen, and M. Nei [http://www.megasoftware.net ]) with corresponding reference EV strains (GenBank accession numbers AY679736, AY208085, AB167989, AM159197, AF160024, AY875692, AM236930, AF295521, AF521340, AF521310, AB055923, DQ092796, AY227344, AF295445, AY208115, AB199314, AJ309245, AJ417364, and AY207635). The phylogenetic trees were built with the MEGA 3.1 program using the neighbor-joining method (34) as implemented in the MEGA computer program. During sequence comparisons, gaps, missing data and ambiguities in the sequences were ignored in pairwise comparisons. In the phylogenetic inference, pairwise genetic distances were calculated by using the Kimura two-parameter model of sequence evolution to account for multiple nucleotide substitutions (27). The reliability of the phylogenetic topologies (branching patterns) was determined by the bootstrap resampling test with 1,000 replicates (15, 27). VP1 sequences obtained from the study are available in the EMBL database under accession numbers AM492321 to AM492504.

Statistical analyses.Quantitative data are presented as means, standard deviation, and range. Qualitative data are presented as the number of observations and percentages. Chi-square tests with or without Yates' correction and analysis of variance tests were carried out when necessary to compare quantitative or qualitative data. The results were considered as statistically significant for two-sided P values of <0.05. The statistical analysis was performed with the SAS software version 8.2 (SAS Institute, Cary, NC).

Virological and clinical features of the study population.Of 11,509 consecutive children enrolled in the study cohort, 2,444 (21%) were positive for the isolation of a human viral strain in their nasopharyngeal sample (Fig. 1A). Of these 2,444 children, 285 (11.6%) demonstrated the presence of an infectious EV strain isolated by classical cell culture assays, with annual rates of EV isolation ranging from 3 to 31% during the 7-year study period (Fig. 1). Of the 285 subjects from which an infectious EV strain had been isolated from a nasopharyngeal aspirate, 252 (88%) were selected because the detection of other common human viral or bacterial pathogens in their nasopharyngeal tracts had remained negative by classical culture or molecular assays at the time of emergency visiting (results not shown). Of these 252 EV-positive children, 111 (44%) had aseptic meningitis, 79 (31%) had upper or lower respiratory tract infections, and 62 (25%) had febrile illness (44% versus 31% versus 25% [chi-square test, P < 10⁻³]; 31% versus 25% [chi-square test, P = 0.11]) (Table 1).

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FIG. 1.

Positive respiratory viral findings in the nasopharyngeal samples of 11.509 consecutive children attending one of the five referent pediatric emergency departments of the region Champagne Ardenne (France) from 1999 through 2005. (A) Distribution (%) of the positive viral isolation of human respiratory strains by classical cell culture assays in nasopharyngeal samples of 2,444 (21%) of 11,509 children during the 7-year of the study period. (B) Distribution of the annual rates of EV-positive isolation by classical cell culture assays among the 2,444 positive nasopharyngeal samples.

Annual and seasonal variations of EV infections.Compared to the total number of the diagnosed EV pediatric infections, the rates of EV-related respiratory syndromes ranged from 5 to 60% during the 7-year study period (Fig. 2). These rates appeared to be significantly lower in years 2000 (chi-square test, P = 0.0002) and 2005 (chi-square test, P = 0.0002), during which epidemics of aseptic meningitis occurred in the region Champagne Ardenne (France) (Fig. 2). From 1999 to 2005, all of the EV-related respiratory infections were diagnosed from February to December. Interestingly, a peak of EV respiratory infection cases occurred in June 2000, 2001, and 2005 and in July 2002. EV respiratory infections had prominent spring-fall seasonality, with June-July period accounting for 47% of all EV infection reports (Fig. 2).

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FIG. 2.

Number of EV-related infections in Champagne Ardenne between 1999 and 2005. *, Years wherein the rates of EV-related respiratory infections appeared to be significantly lower than those of other EV-induced infections (chi-square test stratified according to the years of the study period; P < 0.001).

Virological characteristics of EV strains isolated from the respiratory tract samples of the selected children.Frequencies, ranks, and number of years reported for all of the EV strains isolated from the nasopharyngeal samples of children with respiratory and nonrespiratory tract infections are indicated in the Table 2. Echovirus 11, echovirus 6, echovirus 30, echovirus 13, and coxsackievirus B2 accounted, respectively, for 24, 13, 11, 11, and 11% of all EV respiratory syndromes (Table 2). Echovirus 7, EV 71, coxsackievirus A16, echovirus 11, coxsackievirus B4, coxsackievirus B2, echovirus 3, and coxsackievirus A9 were isolated and identified in 100% (6 of 6 isolates), 100% (1 of 1 isolates), 83% (4 of 5 isolates), 76% (19 of 25 isolates), 66% (4 of 6 isolates), 64% (9 of 14 isolates), 60% (3 of 5 isolates), and 50% (1 of 2 isolates), respectively, of samples from infants with respiratory syndromes, indicating a dominant respiratory tropism for these EV isolates (Table 2).

Phylogenetic comparison of partial VP1 capsid protein region of EV respiratory strains.A phylogenetic tree on partial VP1 sequences of 77 of 79 EV strains isolated from nasopharyngeal samples of children with respiratory pathologies was built, allowing identification of temporal trends and patterns of circulation of respiratory species, serotypes, and strains (Fig. 3). This phylogenetic approach demonstrated the concomitant or successive circulation of species A and B human respiratory EVs within a single French geographical area (Fig. 3). During each year of the study period (1999 to 2005), a cocirculation of genetically distinct EV respiratory serotypes occurred between February and December. Moreover, in the two clusters of strains corresponding to echovirus 7 and echovirus 11 serotypes, we observed the cocirculation of genetically distinct subgroups of strains (bootstrap values of >60%) during the same annual epidemic period (Fig. 3) (22). Finally, for each EV serotype identified as related to both respiratory and neurological pathologies in children, various nucleotide or amino acid phylogenetic analyses of VP1 gene sequences were carried out, and their results did not identify the existence of distinct subgroups of strains that might have temporarily acquired a specific respiratory or neurological tropism (not shown).

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FIG. 3.

Phylogenetic tree based on partial VP1 sequences (348 pb) of 77 human EV isolates identified as the viral cause of the respiratory syndrome diagnosed in French infants between 1999 and 2005. The numbers correspond, respectively, to the month and the year of isolation (set as “month-year” in the tree) of EV strains from pediatric nasopharyngeal samples. Solid boxes indicate the cases of a cocirculation of distinct EV respiratory tropism species during the same month of the same annual epidemic season. Dotted rectangles indicate examples of cocirculation of distinct EV respiratory serotypes within the same species during the same month of the same annual epidemic season. Bootstraps in boldface indicate examples of cocirculation of phylogenetically distinct subgroups belonging to the same EV strain (bootstraps > 60%) during the same month of the same annual epidemic season.

Clinical features of EV-induced respiratory infections.The signs and symptoms of the 79 children with different EV-induced respiratory infections are reported in the Table 3. Three patients demonstrated a secondary bacterial respiratory infection developed during hospitalization and inducing a fatal cardiorespiratory distress in one case (Table 3). Among the 79 children with a documented EV respiratory infection, 43 (54%) had from LRTIs and 36 (46%) had upper respiratory tract infections (54 versus 46%; chi-square test, P = 0.27). During the study period, we observed that bronchiolitis was the most frequent EV-related respiratory pathology diagnosed (43% of 79 cases) compared to flu-like illness (21% of cases), sore throat (11% of cases), or asthma attack (8% of cases) (43 versus 21 versus 11 versus 8%; chi-square test, P < 10⁻³) (Table 3). EV-induced bronchiolitis occurred significantly more often in infants aged 1 to 12 months than in children in other age groups (73.5 versus 26.5%; chi-square test with Yates' correction, P = 0.0002). No EV serotype could be associated with a specific respiratory clinical manifestation (analysis of variance test, P > 0.5).