Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014–2021

To determine molecular epidemiology and clinical features of enterovirus D68 (EV-D68) infections, we reviewed EV-D68–associated respiratory cases at a hospital in Barcelona, Spain, during 2014–2021. Respiratory samples were collected from hospitalized patients or outpatients with symptoms of acute respiratory tract infection or suggestive of enterovirus infection. Enterovirus detection was performed by real-time multiplex reverse transcription PCR and characterization by phylogenetic analysis of the partial viral protein 1 coding region sequences. From 184 patients with EV-D68 infection, circulating subclades were B3 (80%), D1 (17%), B2 (1%), and A (<1%); clade proportions shifted over time. EV-D68 was detected mostly in children (86%) and biennially (2016, 2018, 2021). In patients <16 years of age, the most common sign/symptom was lower respiratory tract infection, for which 11.8% required pediatric intensive care unit admission and 2.3% required invasive mechanical ventilation; neurologic complications developed in 1. The potential neurotropism indicates that enterovirus surveillance should be mandatory.

Regarding the clinical definitions used, upper respiratory tract infections (URTIs) were infections from the nose to the larynx; LRTIs were recurrent wheezing, asthma, bronchiolitis, and pneumonia. To ensure that the length of hospital stay or respiratory support were associated only with EV-D68, we studied LRTI severity in patients requiring admission because of respiratory tract infection. Respiratory support was divided into 5 groups: none, oxygen through nasal cannula, high-flow nasal cannula, noninvasive mechanical ventilation, and invasive mechanical ventilation. EV-D68-associated AFP was defined as myelitis causing sudden onset of paralysis with T2 hyperintensity in medulla gray matter with dorsal brain stem variably affected on magnetic resonance images and EV-D68 detected in respiratory specimens.

Enterovirus Detection and Characterization
We performed enterovirus detection by using specific real-time multiplex reverse transcription, as previously described (8). The characterization of enterovirus was performed by the phylogenetic analyses of the partial viral protein 1 (VP1) coding-region according to the protocol recommended by the World Health Organization, with minor modifications (8).

Statistical Analyses
We performed statistical analysis by using SPSS version 22 (SPSS Inc., https://www.ibm.com). To assess associations between categorical variables, we performed χ 2 testing and calculated Z scores. We considered p<0.05 to be significant.

Discussion
Interest in EV-D68 was limited until the large outbreak that occurred in the United States in 2014 (6,9). Although EV-D68 circulation had been previously described, that large outbreak affecting mainly children was associated not only with severe respiratory disease but also with neurologic complications in some cases. Furthermore, the circulating EV-D68 strains belonged to previously circulating lineages, and therefore, there was no clear evidence of a new virus strain associated with increased severity (6,9). Nevertheless, during the same period, further studies began not only in the United States but also in Europe to monitor EV-D68 circulation (10). Results revealed a low level of EV-D68 detection and milder clinical manifestations in Europe compared with those in the United States (10). Similarly, the EV-D68 circulation in Barcelona was low during that period (8). However, in the following seasons, the trend increased, particularly during 2016 and 2018, as reported in other regions of Spain (11) and Europe (12,13), especially the upsurge observed during the 2021-22 season (7). According to those data, EV-D68 seems to follow a biennial circulation pattern as recently defined (14,15), which was displaced during 2020 because of the CO-VID-19 pandemic, but higher numbers of cases were detected during 2021 (7). Four distinct clades of EV-D68 (A-D) have been described (16) in addition to subclades A, B1, B2, and B3 (10,17). Clades cocirculated variably; B3 predominated during the studied seasons, which is in concordance with other reports (10,18). Moreover, in our study, viruses belonging to the new emerging subclade D1 within clade D, were mainly  (12,19). Furthermore, subclade D1 was observed similarly among pediatric and adult populations, compared with B3, which was mostly detected in children. This age effect depending on clade, in concordance with our results, has been reported in other studies (19,20). Of note, a recent study reported changes in the VP1 region of D1 associated with lower cross-protection in adults (21). The most common clinical features of EV-D68 infection in children in this study were respiratory, and AFP developed in only 1 patient. EV-D68 has been mostly associated with LRTI in children >2 years of age, 50%-70% of whom previously had asthma or recurrent wheezing and 20% of whom had no comorbidities, as described in our study. In addition, most cases from our study were mild; few patients were admitted to intensive care units (ICUs). However, during the US outbreak in 2014, respiratory signs/symptoms reported for hospitalized patients were more severe: 59% of patients required ICU admission, 23% noninvasive mechanical ventilation, and 8% invasive mechanical ventilation; therefore, previous asthma or reactive airway disease might increase the risk for ICU admission and the need for ventilatory support (22). Despite changes in the virologic properties of circulating viruses, the clinical features remained similar to those reported in 2014 in Europe, in contrast to the United States and Canada. In addition, during the US outbreak, AFP cases associated with EV-D68 infection (subclade B3), in which this virus was the only pathogen isolated, increased (6,9). Although enteroviruses are known to be neurotropic, we detected only 1 case of AFP associated with EV-D68 subclade B3 infection.
The potential neurotropism of EV-D68 and other enteroviruses suggests that surveillance should be mandatory, which is one of the aims of the European Non-Polio Enterovirus Network (https:// www.escv.eu/enpen). The year-round circulation of EV-D68 should help with close monitoring of this enterovirus, as well as prompt response to the potential occurrence of outbreaks and related clinical burden.