Enterovirus (EV) D68 was first isolated in 1962 in California, in 4 children with severe acute respiratory infection (ARI).1 For almost 50 years, it was one of the rarest enteroviruses identified in humans.2 However, in 2006, clusters and outbreaks of ARI caused by EV-D68 began to be reported mainly in Asia, Europe and the US.2–4 In 2014, national outbreaks were noted in US and Canada, in which hundreds of children with severe ARI were hospitalized, and required support from pediatric intensive care units (PICUs).2,5,6 In other countries, outbreaks of ARI due to EV-D68 were observed simultaneously, suggesting the global spread of this virus.2,7 Coinciding with these outbreaks an increase in cases of acute flaccid myelitis (AFM) was observed.8,9
The genus Enterovirus consists of naked, single-stranded, positive-sense, RNA viruses that are classified into 15 species of which 7, enterovirus A to D and rhinovirus A to C include viruses that infect humans.10 These infections are frequently asymptomatic, but they are also associated with clinical syndromes that range from mild diseases (eg, febrile rash illness, hand-foot-mouth syndrome, conjunctivitis and herpangina) to severe diseases (eg, aseptic meningitis, flaccid paralysis and myocarditis). EV-D68, until 2002 also known as rhinovirus 87, shares biologic, physiochemical and molecular features with both enteroviruses and rhinoviruses11,12 and it has been associated especially with respiratory tract infections.2
Because of the increase in detections of EV-D68 in severe ARI cases in 2014 and its possible association with AFM, surveillance of the circulation of this virus has been recommended.2,6,7,13 However, there are no commercial molecular diagnostic methods that specifically detect EV-D68, and most of them identify but cannot differentiate between enteroviruses and rhinoviruses. Therefore, the surveillance of EV-D68 requires additional efforts from laboratories. The main objectives of this study were to describe the clinical and virologic results of the passive surveillance of EV-D68 in Gipuzkoa during 2016, and the differences observed when compared with respiratory infections associated to EV non-D68.
MATERIAL AND METHODS
Study Population and Patient Data
This cross-sectional observational study was undertaken in the health areas of San Sebastián, Bidasoa, Goierri and Tolosa (Gipuzkoa) in the Basque Country, northern Spain, with a population of 518,572 inhabitants, of which 75,282 were less than 15 years old (2016 census). The Donostia University Hospital (77 pediatric beds, reference hospital) and the Hospital of Zumarraga (10 pediatric beds), are the public hospitals in this region and cater for more than 90% of pediatric hospitalizations.
The presence of RNA/DNA of respiratory viruses, including enteroviruses, was investigated in all respiratory samples of children less than 15 years of age hospitalized with ARI. In the case of children with ARI attended in the Pediatric Emergency Care Units, respiratory samples were investigated at the clinician discretion. From January 2015 to December 2017, in all enterovirus-positive respiratory samples, the presence of EV-D68 RNA was investigated. In addition, we investigated the presence of RNA of EV-D68 in all cerebrospinal fluids (CSFs) obtained during 2016 that had a positive result for enterovirus. Enterovirus-positive samples were kept frozen at –80°C until subsequent analysis. Clinical records from patients infected with enteroviruses during 2016 were reviewed by 2 pediatricians, and demographic and clinical data were gathered.
Enterovirus and EV-D68 Detection
Respiratory samples were nasopharyngeal washes in children younger than 5 years old, while nasopharyngeal washes or oropharyngeal swabs were collected in children 5–14 years old. Viral DNA and RNA were automatically extracted using the NucliSENS EasyMag (BioMérieux, Marcy l’Etoile, France). A commercial one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) assay, which detects and identifies 18 viruses including enterovirus and rhinovirus separately (Allplex Respiratory Panel; Seegene Inc., Seoul, Republic of Korea) was used. In CSF, enterovirus was detected using a commercial RT-PCR assay (Xpert EV, GenXpert; Cepheid, Sunnyvale, CA). To detect EV-D68, a specific PCR that amplifies a fragment of 965 bp of the viral polyprotein (VP1) gene of this virus was performed in the enterovirus-positive samples.14 Transcription of RNA to complementary DNA was previously performed with M-MuLV reverse transcriptase (Promega, Madison, WI) using random primers. The obtained amplicons were sequenced in an ABIPRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences obtained were analyzed at the Basic Local Alignment Search Tool (BLAST) website (http://www.ncbi.nlm.nih.gov/blast) and in the “RIVM Enterovirus Genotyping Tool” Version 1.0 http://www.rivm.nl/mpf/enterovirus/typingtool. The phylogenetic study of the partial VP1 gene was conducted in Mega-6 through the maximum-likelihood method using the best model, Kimura 2-parameter+G, as determined also in Mega 6 using the Bayesian information criterion, with 1000 bootstrap replications for branch support.15 Sequences of EV-D68 detected in this study were deposited in GenBank (accession numbers KX530786, KX530787 and MH307365-MH307407).
The χ2 test was applied to compare categoric variables with application of Fisher corrections (2 tailed) when required. The Mann-Whitney U test or analysis of variance was used to compare the means of continuous variables. A P value of <0.05 was considered statistically significant. Incidences and 95% exact binomial confidence intervals (CIs) were calculated using the official 2016 census of the Basque Institute of Statistics (http://en.eustat.eus/indice.html) (Supplementary file 1, http://links.lww.com/INF/D424). The database with patient data was anonymized (nameless) so that no child could be identified. The study was approved by the Ethics Committee for Clinical Research of the Donostia University Hospital (Act 11/2017).
During January to December 2016, 2108 respiratory samples, 2007 nasopharyngeal washes (95.2%) and 101 oropharyngeal swabs (4.8%), from 1753 children were analyzed. EV-D68 was detected in 45 respiratory samples of 44 patients, which accounted for 19.8% of patients in which enterovirus was detected (n = 222) and 2.1% of the total samples analyzed. The remaining 178 enteroviruses were classified as EV non-D68. The first EV-D68 was detected on February 2016, and since then it was detected every month, with March, May and June being the months with the highest incidence (Fig. 1). The median age of these patients was 30.1 months (range 1 month–13 years), and 21 (47.7%) were males. EV-D68 was detected in 23 (52.3%) children less than 2 years of age, in 15 children (34.1%) 2 to < 5 years old and in 6 children (13.6%) between the ages of 5 to <15 years. In 2015, 69 of 137 enterovirus-positive respiratory samples had remnant material to perform the EV-D68 specific PCR, and this virus was only detected in 1 sample from December (a 55-day-old male with apnea that did not required hospitalization). During 2017, in the 89 enterovirus-positive respiratory samples analyzed, the specific EV-D68 PCR yielded a negative result in all of them. Finally, in all 247 CSF enterovirus-positive samples analyzed during 2016, the investigation for EV-D68 yielded a negative result.
The illness of 37 (84.1%) cases infected with EV-D68 was manifested as ARI, with increased work of breathing in 30 (68.2%), wheezing in 28 (63.6%) and hypoxia in 24 (54.5%). Five patients received the main diagnosis of unspecific febrile syndrome (11.4%) and 2 patients presented apnea and AFM, respectively (Supplementary table 1, http://links.lww.com/INF/D425), together with mild upper respiratory disease. About half of patients (n = 24) presented with underlying conditions among which a previous history of asthma in 12 cases (27.3%), and recurrent wheezing in 5 (11.4%), stood out. A chest radiograph was performed in 31 patients, and perihilar thickening was the most common radiologic pattern (41.9%) followed by consolidation/infiltration (29.0%). Hospital admission was required in 32 (72.7%) EV-D68 positive patients (14 under 2 years of age, 13 of 2 to <5 years of age and 5 of 5 to <15 years of age) and 7 of them (21.9%) were admitted to the PICU. All hospitalized children received respiratory support. Four patients (9.1%) had complications at 6 months of the discharge; 2 had recurrent bronchitis, the child with AFM presented with limb weakness and a patient with Taussig-Bing heart disease died during the infection.
Coinfections were detected in 8 children (18.2%) of which 6 were viral [rhinovirus, n = 4; influenza A H1N1 pandemic, n = 1 and respiratory syncytial virus (RSV), n = 1]. In addition, 3 children had a bacterial coinfection (one of these simultaneously to RSV infection) (Supplementary table 2, http://links.lww.com/INF/D426). Among the 178 children infected with EV non-D68, 69.7% (n = 124) had a coinfection (P < 0.001), being the most frequent infections by rhinovirus (n = 46), influenza virus (n = 32) and RSV (n = 19).
When the main clinical and epidemiologic characteristics of children with respiratory infection associated with EV-D68 versus EV non-D68 during 2016 were compared, in which no coinfections were detected (36 and 54 children, respectively), several differences were observed (Table 1). Children with EV-D68 infection presented more frequently with asthma as underlying condition. Also, during the infection, increased work of breathing, wheezing and hypoxia were more common, and chest radiographs had pathologic images more frequently. On the other hand, hospitalization was more frequent among children with EV-D68 infection and also these children required more respiratory support, corticosteroids and bronchodilators as treatment than children infected with EV non-D68. Among the hospitalized children, asthmatic attack and recurrent wheezing were more common as discharge diagnosis.
The estimated annual incidence rates of hospital admission due to community-acquired EV-D68 infection per 10,000 inhabitants in 2016 were 17.0 (95% CI: 10.2–28.5), 9.9 (95% CI: 5.8–16.9) and 1.1 (95% CI: 0.5–2.5) for patients less than 2, 2 to <5 and 5 to < 15 years old, respectively (Supplementary file 1, http://links.lww.com/INF/D424).
All 44 EV-D68 detected in Gipuzkoa during 2016 and the strain detected in 2015 could be typed and belonged to lineage B3 (Fig. 2). Nucleotide sequence identities of the amplified fragment in the VP1 gene was 97.2–100% among the Gipuzkoa strains, 9 of them being identical. The strain SS/70120874 (arbitrarily selected among the 9 identical strains as prototype of this cluster of strains) showed 97.4–98.4% of nucleotide identity with strains of lineage B3 that circulated in China and Taiwan in 2013 and 2014 (GenBank accession numbers KT853090 and KT711088 KT853091), 98.9–99.0% with the strains detected in China in 2015 (KU952558-59) and 99.0–99.1% with the strains detected in US in 2016 (KX957758, KX957759, KX675261 and KX675263), being very similar (>99.3%) to other strains isolated in Spain in 2016 (KX949561 and KX949563).
The present study describes a period of intense circulation of EV-D68 of the recently described lineage B3 in Gipuzkoa (Basque Country, Spain) in 2016, which was associated with ARI cases in children, including several episodes of hospitalization, mainly due to difficulty breathing and hypoxemia. The annual incidence rate of hospitalization [11.7/10,000 inhabitants < 5 years old (95% CI: 8.1–17.0)] was higher than that observed in a prospective study performed in Canada during the EV-D68 upsurge of 2014 (2.1/10,000 for the period 28 August to 31 December).16 The annual rate obtained in children < 3 years old [16.2/10,000 inhabitants (95% CI: 10.6–25.1)] was close to those previously referred in Gipuzkoa (July 2004 to June 2007) for important respiratory viruses like human metapneumovirus (26/10,000), parainfluenza virus (17/10,000) and influenza (10/10,000).17
Underlying medical conditions were common in patients with EV-D68 infection, especially asthma and recurrent wheezing.5,18–21 These illnesses were also the most frequent clinical presentations of acute EV-D68 infection, like in other series that studied hospitalized children. The diagnosis most frequently associated with EV-D68 infection was recurrent wheezing in Spain in 2012–2013,14 respiratory distress, upper respiratory tract infection and wheezing in US in 2011–2014,20 and status asthmaticus or asthma exacerbation in the Netherlands in 2016.22 Moreover, a nonspecific respiratory disease, indistinguishable of asthma exacerbation related to other causes, was the predominant presentation in hospitalized patients, mostly children, during the nationwide outbreak of EV-D68 that occurred in the US in 2014.5 In Japan, the increase in pediatric asthma hospitalizations observed in September 2015 was found to be associated with an EV-D68 epidemic.23
EV-D68 hospitalized children (21.9%) needed treatment in the PICU, for close monitoring and respiratory support. This proportion was in line with the rates of 8–20% referenced in some large studies,16,18,19 but was significantly lower than that reported in others.5,21 The variation in these data, like that observed in relation to the proportion of inpatients,5,18 are a consequence partly of the design of the studies, frequently hospital-based passive surveillance studies that are more sensitive to severe disease.16 This problem is aggravated because of the lack of commercial assays for the diagnosis of EV-D68, and therefore the need to use laboratory-developed assays, which frequently are performed only in patients with severe infections. In addition, there are differences in the hospitalization criteria and clinical practices followed at different regions. However, taken together, the studies indicate that during periods of EV-D68 epidemic circulation, severe respiratory infections caused by this virus are common, and that a history of asthma and other comorbidities are potential risk factors for severe disease.20
When the characteristics of the infections by EV-D68 and by EV non-D68 were compared, several differences emerged, in agreement with the results of previous studies.19,20 Children with EV-D68 infection had a history of asthma and during acute infection they presented with increased work of breathing more frequently. Clinical outcomes severity indicators (hospitalization, respiratory support) were more frequent in children with EV-D68 infection as well as treatment with corticosteroids and bronchodilators. Recurrent wheezing and asthmatic crisis were the most frequent discharge diagnosis in children hospitalized with acute EV-D68 infection, in contrast to the infections in which EV non-D68 were detected, which presented more varied diagnoses, being the most frequent nonspecific febrile syndrome. Viral and bacterial coinfections were rarely detected in the context of infections by EV-D68 (18.2%), but were frequent in infections by EV non-D68 (69.7%), especially with influenza virus and RSV. Probably the intensive surveillance of respiratory viruses by molecular multiplex techniques favored the detection of EV non-D68 in cases of ARI in which they probably did not play a relevant pathogenic role.
Over the past 2 decades, multiple clades of EV-D68 have emerged and disseminated worldwide.24 All the strains detected in the present study were assigned to clade B, lineage 3 (lineage B3). This new lineage emerged in 2013 in China21,25 and circulated widely in China and Taiwan in 2014,21,26 where it was associated with severe infection. In 2016, outbreaks and severe cases caused by closely related strains of B3 lineage, were identified in the US21 and some European countries,23,27–30 including the intense circulation period described in the present study. The American and European strains were more closely related to strains detected in Southern China and Japan in 2015.21,31 These data indicate that the EV-D68 lineage B3 has spread internationally during 2016, possibly at the global level, as it happened in 2014 with strains of lineage B1.5,8,18,21,32
There were some limitations to this study. It was conducted from 2 hospitals so it was not possible to properly analyze the infections in outpatients, and therefore to know the global impact of the EV-D68 circulation on the Health System, since the study did not include children treated in Primary Care, nor all those treated in the “Emergency room” with ARI. Moreover, it was a passive surveillance study, and a case-definition was not implemented, so it may have missed cases. On the other hand, we do not know the genotypes of EV non-D68 that circulated during the study period and neither if the comparative results between EV-D68 and non-D68 would be reproducible in other periods, given the genetic diversity of enteroviruses. It has been reported that previous versions of the commercial PCR used in our laboratory to detect enteroviruses in respiratory samples, Anyplex IIRV16 and Seeplex RV15 Ace One-step assays (Seegene, Seoul, South Korea), failed to detect EV-D68.18,33 However, using the new version Seegene Allplex Respiratory panel kit, we detected a large number of infections caused by this virus. Finally, we cannot definitively rule out that cases of coinfection between EV-D68 and rhinovirus correspond to cross-reactions, given that we did not have a remaining sample to perform monoplex PCR and sequencing. However, it has been reported that the previous version of the molecular method used accurately differentiated between rhinovirus and enterovirus.34
In conclusion, the period of intense circulation of EV-D68 lineage B3 in Gipuzkoa (Basque Country, Spain) in 2016, had an important impact in the pediatric population and was associated to ARI, with high incidence of hospitalization and admission in the PICU. It would be convenient to strength the surveillance systems of EV-D68 circulation, which would be facilitated by the development of commercial molecular methods to identify this virus.
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