Research Molecular epidemiology of an enterovirus A71 outbreak associated with severe neurological disease, Spain, 2016 Rubén González-Sanz¹, Didac Casas-Alba², Cristian Launes2,20, Carmen Muñoz-Almagro2,19,20, María Montserrat Ruiz-García³, Mercedes Alonso⁴, María José González-Abad⁴, Gregoria Megías⁵, Nuria Rabella6, Margarita del Cuerpo6, Mónica Gozalo- Margüello7, Alejandro González-Praetorius⁸, Ana Martínez-Sapiña⁹, María José Goyanes-Galán10, María Pilar Romero11,21, Cristina Calvo11,21, Andrés Antón12, Manuel Imaz13, Maitane Aranzamendi14 , Águeda Hernández-Rodríguez15, Antonio Moreno-Docón16, Sonia Rey-Cao17, Ana Navascués18, Almudena Otero1,21, María Cabrerizo1,20,21 1. Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain 2. Institut de Recerca Sant Joan de Déu, Barcelona, Spain 3. Hospital General de Elche, Alicante, Spain 4. Hospital Infantil Universitario Niño Jesús, Madrid, Spain 5. Complejo Hospitalario de Burgos, Burgos, Spain 6. Hospital Santa Creu i Sant Pau, Barcelona, Spain 7. Hospital Universitario Marqués de Valdecilla, Santander, Spain 8. Hospital Universitario de Guadalajara, Guadalajara, Spain 9. Hospital Miguel Servet, Zaragoza, Spain 10. Hospital Gregorio Marañón, Madrid, Spain 11. Hospital Universitario La Paz, Fundación IdiPaz, Madrid, Spain 12. Hospital Universitari Vall d´Hebron, Barcelona, Spain 13. Hospital de Basurto, Bilbao, Spain 14. Hospital de Cruces, Bilbao, Spain 15. Microbiology Service, University Hospital “Germans Trias i Pujol”, Department of Genetics and Microbiology, Autonomous University of Barcelona, Badalona, Spain 16. Hospital Universitario Virgen de la Arrixaca, Murcia, Spain 17. Hospital General de Vigo, Vigo, Spain 18. Complejo Hospitalario de Navarra, Pamplona, Spain 19. Universitat Internacional de Catalunya, Barcelona, Spain 20. CIBER de epidemiología y Salud Pública, CIBERESP, Madrid, Spain 21. Translational Research Network in Paediatric Infectious Diseases (RITIP), IdiPaz, Madrid, Spain Correspondence: Rubén González-Sanz (ruben.gs@isciii.es) Citation style for this article: González-Sanz Rubén, Casas-Alba Didac, Launes Cristian, Muñoz-Almagro Carmen, Ruiz-García María Montserrat, Alonso Mercedes, González-Abad María José , Megías Gregoria, Rabella Nuria, del Cuerpo Margarita, Gozalo-Margüello Mónica, González-Praetorius Alejandro, Martínez-Sapiña Ana, Goyanes-Galán María José, Romero María Pilar, Calvo Cristina, Antón Andrés, Imaz Manuel, Aranzamendi Maitane, Hernández-Rodríguez Águeda, Moreno-Docón Antonio, Rey-Cao Sonia , Navascués Ana, Otero Almudena, Cabrerizo María. Molecular epidemiology of an enterovirus A71 outbreak associated with severe neurological disease, Spain, 2016. Euro Surveill. 2019;24(7):pii=1800089. https://doi.org/10.2807/1560-7917.ES.2019.24.7.1800089 Article submitted on 27 Feb 2018 / accepted on 09 Jul 2018 / published on 14 Feb 2019 Introduction: Enterovirus A71 (EV-A71) is an emerging and July 2016 (83%). Most isolates (218/233) were pathogen that causes a wide range of disorders classified as subgenogroup C1 and 217 of them were including severe neurological manifestations. In the grouped in one cluster phylogenetically related to a past 20 years, this virus has been associated with new recombinant variant strain associated with severe large outbreaks of hand, foot and mouth disease neurological diseases in Germany and France in 2015 with neurological complications in the Asia-Pacific and 2016. Moreover, we found a clear association of region, while in Europe mainly sporadic cases have EV-A71-C1 infection with severe neurological disorders, been reported. In spring 2016, however, an EV-A71 brainstem encephalitis being the most commonly outbreak associated with severe neurological cases reported. Conclusion: An emerging recombinant was reported in Catalonia and spread further to other variant of EV-A71-C1 was responsible for the large Spanish regions. Aim: Our objective was to investigate outbreak in 2016 in Spain that was associated with the epidemiology and clinical characteristics of the many severe neurological cases. outbreak. Methods: We carried out a retrospective study which included 233 EV-A71-positive samples collected Introduction during 2016 from hospitalised patients. We analysed Enterovirus 71 (EV-A71) is a small, non-enveloped, the clinical manifestations associated with EV-A71 single-stranded RNA virus that belongs to the species infections and performed phylogenetic analyses of the Enterovirus A along with 24 other serotypes within 3’-VP1 and 3Dpol regions from all Spanish strains and the Enterovirus genus [1]. According to the VP1 protein a set of EV-A71 from other countries. Results: Most sequence, EV-A71 is classified into six genogroups EV-A71 infections were reported in children (mean age: (A–F) and a number of subgenogroups (B0–B5, C1–C5) 2.6 years) and the highest incidence was between May [2]. Although EV-A71 infection is often asymptomatic, www.eurosurveillance.org 1 Figure 1 it can cause disorders with a wide range of clinical Phylogenetic analysis of 3´-VP1 sequences of manifestations from non-specific febrile illness, enterovirus-A71, Spain, 2016 (n = 233) and representatives of different genogroups worldwide aseptic meningitis and mild mucocutaneous symptoms to severe neurological diseases such as brainstem encephalitis and acute flaccid paralysis (AFP) [3,4]. EV-A71 is distributed worldwide. However, the largest outbreaks associated with hand, foot and mouth disease (HFMD) with subsequent neurological and cardiopulmonary complications have been described in the Asia-Pacific region, especially in the past 20 years [2,5-7]. These outbreaks have been connected to the 95 C1-variant circulation of different subgenogroups (B3, B4, C1, C2 and C4) [8-14]. In Europe, although outbreaks of polio- like disease occurred in Hungary and Bulgaria in the 1970s [15,16], only sporadic cases have been reported C1 from several countries in recent years, mainly caused by the C1 and C2 subgenogroups [2,17]. In 2015, a new recombinant EV-A71 variant was identified 94 AB575938 Netherlands 2010 KU641487 Germany 2011 that affected at least 19 young children in different areas of Germany [18]. This infection was associated KU641488 Germany 2013 94 KU641489 Germany 2013 JQ973699 Russia 2007 FN598766 France 2006 with neurological manifestations (cerebral seizures, HG934162 Germany 2003 71 HQ676156 Spain 2002 DQ452074 Norway 2003 myoclonia and ataxia) that required hospitalisation. HQ676159 Spain 2003 H AM939605 UK 2006 There were no reports of fatal cases or clinical sequelae 07527 SP FEB16 M HQ676158 Spain 2002 C after hospital discharge. Moreover, a well-documented AB524169 Netherlands 2005 AB524187 Netherlands 2007 case, a 2-year-old girl, required hospitalisation and HQ676157 Spain 2002 FN598746 France 2006 was diagnosed with brainstem encephalitis and FN649268 Austria 2003 EU527983 Taiwan 2007 C5 cardiopulmonary complications with an outcome of KU159482 Vietnam 2012 a probable persistent neurological impairment [19]. 99 KC688862 Spain 2012 KC688859 Spain 2011 KC688860 Spain 2012 KC688863 Spain 2012 In addition, 18 cases of severe neurological disease 56965 SP MAY16 Z HG934296 France 2013 associated with EV-A71 infection, and phylogenetically 99 35991 SP JUN16 MU 35278 SP JUN16 MU closely related to the strains described in Germany, KC688861 Spain 2012 35269 SP JUN16 MU were reported in France between May and October KC688864 Spain 2012 47070 SP JUN16 B 2016. Patients presented with rhombencephalitis, HQ676288 Russia 2009 KC688865 Spain 2012 encephalitis or encephalomyelitis, and one fatal case of HG934163 France 2010 99 HE573011 France 2010 HE573009 France 2010 C2 acute cardiac failure was reported [20]. The same strain 97 HQ676160 Spain 2007 FR798002 Spain 2008 was also involved in a sporadic case of encephalitis in 81 HG934164 France 2010 HQ676273 Netherlands 2007 Poland during summer 2016 [21]. HQ676271 Netherlands 2007 95 20787 SP APR16 AV 86 99 20785 SP APR16 AV 36986 SP JUL16 CC In Spain, EV-A71 was circulating at a very low rate until 2015 [22,23]. In the spring of 2016, however, a large 35702 SP JUN16 CC 35690 SP JUL16 A 35674 SP JUL16 A 100 24497 SP MAY16 B 26229 SP MAY16 B outbreak associated with severe neurological diseases 97 53648 SP JUN16 B 26287 SP APR16 B was reported in the region of Catalonia [24-26] and DQ341355 South Korea 2000 JQ973705 Russia 2011 C3 further disseminated to the rest of the country. KX267855 Taiwan 2011 100 KF900160 France 2012 C4 FJ158600 China 2008 96 GU198376 China 2008 In the present study, we investigated the clinical A EV-71 BrCr AY179600 India 2001 D manifestations of EV-A71 infections and the molecular HG421068 Central African Republic 2008 E HG421068 Madagascar 2004 F epidemiology and geographical spread of the strains HG421069 Madagascar 2011 detected in Spain in 2016, comparing them to strains 97 99 FJ357371 Taiwan 1986 B1 JN874548 Taiwan 1986 89 FJ357361 Taiwan 2008 KR068480 Malaysia 2013 B5 circulating in Spain and other countries in recent years. 83 AB524122 Netherlands 1986 B2 B U22522 USA 1987 98 AF376117 Singapore 1998 JN874550 Malaysia 1997 B3 Methods 78 FJ357365 Taiwan 2001 B4 98 HuJF420548 Taiwan 2006 Coxsackievirus A16 G-10 Enterovirus surveillance system 0.05 The Spanish National Reference Laboratory for A: Alicante; AV: Ávila; B: Barcelona; CC: Cáceres; EV: enterovirus; M: Madrid; MU: Enterovirus (SNRLE) conducts the EV surveillance Murcia; Z: Zaragoza. system at a national level. The system is voluntary MEGA 7.0 software was used to construct the neighbour-joining and maximum composite likelihood tree. Bootstrap values > 70% are indicated in the branch nodes. and EV-positive clinical samples are received for CV-A16 prototype strain G-10 was used as the outgroup. Sequences are labelled with their GenBank accession number, country and year of isolation. Spanish sequences characterisation from patients of all ages admitted from 2016 are represented by black circles and labelled with identification number, country abbreviation, month and year of isolation and province. to hospitals throughout the country. Each specimen 2 www.eurosurveillance.org Figure 2 Phylogenetic analysis of 3Dpol sequences of enterovirus-A71, Spain, 2016 (n = 187) and representatives of different members of Enterovirus A species 76 C1 variant KP289442 Coxsackievirus A4 China 2013 KY271949 Coxsackievirus A4 USA 2015 99 HQ423142 EV-A71 China 2009 KC878551 EV71 China 2012 KP144346 Coxsackievirus A6 Finland 2008 KX212498 Coxsackievirus A6 Thailand 2009 JQ946055 Coxsackievirus A6 Taiwan 2010 70 99 KP289695 Coxsackievirus A6 China 2013 KP289920 Coxsackievirus A6 China 2013 KP289928 Coxsackievirus A6 China 2013 83 KM215267 Coxsackievirus A16 China 2014 KM516102 Coxsackievirus A16 China 2010 86 JQ746678 Coxsackievirus A16 Malaysia 2006 86 KP266573 Coxsackievirus A16 China 2008 U22521 EV71 BrCr DQ34155 EV71 South korea 2000 90 HQ676289 EV71 Spain 2002 HQ676292 EV71 Spain 2003 HQ676320 EV71 Iceland 2004 HQ676471 EV71 Netherlands 2007 AB524310 EV71 Netherlands 2001 AM939621 EV71 UK 1998 88 78 HQ676296 EV71 Finland 2000 07527 SP FEB16 M 95 HQ676291 EV71 Spain 2002 72 HQ676290 EV71 Spain 2003 AM939609 EV71 UK 2006 DQ452074 EV71 Norway 2003 7998 HQ676294 EV71 Spain 1999 HQ676295 EV71 Spain 1999 56965 SP MAY16 Z 47070 SP JUN16 B 35269 SP JUN16 MU 26229 SP MAY16 B 93 26287 SP APR16 B 24497 SP MAY16 B 53648 SP JUN16 B EV-A71-C1-C2 KY888025 EV71 USA 2016 35674 SP JUL16 A 98 35690 SP JUL16 A 96 35278 SP JUN16 MU 35991 SP JUN16 MU 99 20785 SP APR16 AV 20787 SP APR16 AV HHQ676316 EV71 UK 2010 HQ676314 EV71 UK 2010 HQ676312 EV71 UK 2010 AM939608 EV71 UK 2006 HQ647167 EV71 Canada 2007 HQ676481 EV71 Netherlands 2010 HQ676487 EV71 Russia 2009 FJ172159 EV71 Singapore 2008 HQ676293 EV71 Spain 2007 HQ676301 EV71 Finland 2007 AB524327 EV71 Netherlands 2007 HQ676470 EV71 Netherlands 2007 HQ676467 EV71 Netherlands 2007 HQ676472 EV71 Netherlands 2007 EF667344 Human enterovirus 92 AY697458 Human enterovirus 76 AY697459 Human enterovirus 89 80 AY697460 Human enterovirus 90 AY697461 Human enterovirus 91 AY426531 EV-D68 0.5 A: Alicante; AV: Ávila; B: Barcelona; EV: enterovirus; M: Madrid; MU: Murcia; Z: Zaragoza. MEGA 7.0 software was used to construct the neighbour-joining and maximum composite likelihood tree. Bootstrap values > 70% are indicated in the branch nodes. EV-D68 prototype strain Fermon was used as the outgroup. Sequences are labelled with the GenBank accession number, country and year of isolation. Spanish sequences from 2016 are represented by black circles and labelled with country abbreviation, month and year of isolation and province. www.eurosurveillance.org 3 is sent with information on patient demographics, belonging to subgenogroups C1 and C2 as well as clinical diagnosis and date of sample collection. For sequences of members of the Enterovirus A species the purposes of this study, more detailed information with which recombination events normally occur. The about the severity of the symptoms, final diagnosis and EV-D68 Fermon prototype strain (AY426531), a member outcome was subsequently collected from hospitals of Enterovirus D species, was included as an outgroup. for patients with neurological disease. EV-positive samples from paediatric patients who were enrolled in The sequences obtained in this study have been a prospective and multicentre project (PI15CIII-0020) deposited in GenBank under accession numbers were also included in the study after informed consent MH394906–MH395138 (VP1 sequences) and from parents or legal guardians. This project includes MH394719–MH394905 (3Dpol sequences). 13 representative hospitals in Spain that perform active surveillance of children with neurological and Statistical analysis systemic diseases associated with EV and parechovirus Significant variations between groups were infections. In addition, they provide us with extended evaluated using the chi-squared test. A difference clinical information about the patients. The procedure with p value < 0.05 was considered to be statistically to collect and send samples to the SNRLE does not significant. differ from the routine EV surveillance system. Results Clinicians defined the severity of the neurological disorders according to their hospital’s protocols. Virological findings For this study, a consensus was reached to define During 2016, the SNRLE received 1,113 EV-positive severe neurological diseases according to clinical samples (mainly CSF, serum, stools and respiratory manifestations; it included encephalitis (encephalitis, samples) from 1,029 patients admitted to different meningoencephalitis and brainstem encephalitis), AFP/ Spanish hospitals with clinical pictures of fever without myelitis, and other motor disorders (ataxia, instability, source (FWS) (n = 174; 16.9%), HFMD/exanthema cerebellitis). Mild neurological disease included (n = 52; 5.1%), respiratory illnesses (n = 270; 26.2%), patients with aseptic meningitis with a good evolution the neurological diseases described above (n = 470; on their own. 45.7%), and others (n = 63; 6.1%). In the case of 84 patients from whom two different positive clinical Enterovirus characterisation and phylogenetic samples were received, only the specimen with the analysis oldest collection date was included in the study. RNA was extracted from clinical samples using the QIAamp Viral RNA Mini Kit (QIAGEN, Germany). EV After discarding duplicates, 777 EV were typed, EV-A71 genotyping was performed using four specific nested being the most common serotype (n = 233; 30.0%). The RT-PCRs for the species EV-A, B, C and D and further following five most frequent serotypes were EV-D68 sequencing of the 3’-VP1 region as previously described (n = 148; 19.0%), echovirus (E)-30 (n = 113; 14.5%), E-5 [27]. In EV-A71-positive samples, the 3Dpol region was (n = 67; 8.6%), CV-A6 (n = 30; 3.8%) and E-7 (n = 23; amplified and sequenced by specific nested RT-PCR as 3.0%). Three EV-A71-positive patients were co-infected previously described [28]. with EV-D68, one with E-7 and one with E-11. We carried out phylogenetic analysis based on 3’-VP1 Epidemiological data of patients with (359 bp) sequences of EV-71-positive samples. In enterovirus A71 addition, we performed 3Dpol (738 bp) analysis to Specimens of EV-A71 were isolated mainly from confirm possible recombination events [28,29]. Multiple respiratory (n = 150, 64.4%) and stool samples (n = 71, sequence alignments were done in the ClustalW 30.5%) but were also detected in cerebrospinal fluid programme. Genetic distances were calculated using (n = 8, 3.4%) and serum (n = 4, 1.7%). Both a respiratory the maximum composite likelihood nucleotide distance and a stool sample were available in 34 EV-A71 cases. model, and statistical significance of phylogenies was EV-A71 was only successfully genotyped from both estimated by bootstrap analysis with 1,000 replicates. samples in 15 of these cases. Patients’ ages ranged Phylogenetic trees were constructed following the from 1 day to 63 years, with a mean of 2.6 years (n = 231 neighbour-joining method using MEGA 7.0 software. with available information on age; standard deviation In the case of 3´VP1, EV-A71 sequences from 2016 (SD) = 5.35; median = 1.86; 95th percentile = 5.9). The were compared with EV-A71 sequences available in male/female sex ratio was 1.5/1. The period from GenBank belonging to the C1 and C2 subgenogroups symptom onset to collection date was only provided in circulating in Spain and other European countries in 36 cases with a mean of 3.3 days (SD = 1.9; median = 3). recent years, along with representative members of the genogroups A, B, C, D, E and F. The coxsackievirus Phylogenetic analysis of enterovirus-A71 (CV) A16 G-10 prototype strain (CAU05876), a member The VP1 phylogenetic analysis revealed that most of Enterovirus A species, was included as an outgroup. of the Spanish sequences from 2016 (218/233) For the phylogenetic analysis of 3Dpol, we compared belonged to subgenogroup C1. Of these, 217 formed EV-A71 sequences from 2016 with EV-A71 sequences a separate group within the C1 cluster together with 4 www.eurosurveillance.org Figure 3 Geographical distribution of enterovirus-A71 cases, Spain, 2016 (n = 233) 0 France 1–2 3–4 5–9 11 6 3 Andorra 10–20 1 21–40 4 1 1 11 >40 3 1 10 (1) 63 (5) 1 Barcelona 2 10 3 (2) 40 (1) Madrid 4 Portugal 13(2) 4 6 6 2 Balearic islands 3 20 (2) (3) Alicante Canary islands 1 The figure shows the total number of EV-A71 cases reported in each province. EV-A71 cases that were not genotyped as EV-A71-C1 variant are shown in brackets. sequences from a new recombinant variant described cases of the outbreak (subgenogroup C1-variant) in Germany in 2015 (bootstrap value > 95%) (Figure 1, were described in Catalonia in April 2016. From May Supplementary Figure S1) [18], while one C1 isolate onwards, the number of cases increased, most of them grouped with EV-A71-C1 sequences from previous concentrated between May and July (Figure 4), and years. The remaining 15 sequences were grouped spread throughout the country. The last two cases within the subgenogroup C2 cluster. were reported in December. A sporadic case associated to subgenogroup C1 but unrelated to the outbreak was Because of insufficient sample volume or unsuccessful reported in February 2016. The cases associated with sequencing, only 187 sequences were available for the subgenogroup C2 were distributed from April to the 3Dpol phylogenetic analysis. Similarly to the VP1 July (Figure 4). analysis, most sequences appeared in one differentiated cluster which again included the sequences from Clinical presentations of enterovirus-A71 Germany in 2015 (bootstrap value > 76%) (Figure 2, infections Supplementary Figure S2). Interestingly, the same Patients involved in the outbreak (EV-A71-C1-variant; sequences that were grouped in the C2 subgenogroup n = 217) were diagnosed with HFMD or herpangina cluster according to the VP1 sequence, were also (n = 8; 3.7%), FWS (n = 14; 6.5%), lower respiratory grouped separately in an interspersed cluster together tract infection (n = 17; 7.8%), neurological symptoms with C1 and C2 subgenogroups sequences. (n = 167; 77.0%) and other symptoms (n = 11; 5.1%) (Table). There was a significantly higher proportion Temporal and geographical distribution of of neurological manifestations with EV-A71-C1-variant enterovirus-A71 infection with severe symptomatology (n = 140; 64.5%) The total of 233 EV-A71-positive samples were collected compared with those with mild symptomatology from 45 different hospitals in 26 of the 50 Spanish (n = 27; 12.4%) (p < 0.0001) (Table). Among the patients provinces and from Andorra. Barcelona and Madrid, infected with EV-A71-C2, five had mild neurological the most populated provinces in Spain, were the two disease, five had severe neurological disease, three with the highest number of cases (Figure 3). The first had lower respiratory tract infection and two had FWS. www.eurosurveillance.org 5 Figure 4 Temporal distribution of enterovirus-A71 cases, by epidemiological week, Spain, 2016 (n = 233) 30 EV-A71-C1 EV-A71-C2 25 EV-A71-C1 unrelated to the outbreak Number of cases 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Epidemiological weeks Data are represented according to the specimen collection date. The remaining non-outbreak-related EV-A71-C1 case in Europe compared with the Asia-Pacific region, and was reported as having FWS (Table). most documented EV-A71 infections included sporadic According to the data provided by hospitals, 22.9% cases of febrile illness, HFMD and meningitis [5,6]. (33/144) of the children with severe neurological However, several cases of EV-A71 infections associated diseases required admission to a paediatric intensive with severe neurological disorders in children were care unit and 65.3% of them (94/144) received some reported in Germany and France during 2015 and 2016 treatment (intravenous gamma globulin or/and [18-20]. In Spain, an increasing number of EV-A71 corticoids). After 90 days of follow-up, 88.2% (n = 127) infections was noticed in 2016 compared with previous of them had no significant sequelae. years. From 2000 to 2015, the SNLRE had identified an average of two isolates of EV-A71 per year with a To confirm the association of EV-A71-C1-variant maximum of seven cases in 2012 [22,23,30]. infection with severity, we analysed the total of severe neurological infections positive for EV during 2016 Regarding specimen types, EV-A71 was mainly detected (n = 190). Meningoencephalitis was the most common in either respiratory samples or stools (or both). This is clinical manifestation (n = 79; 41.6%) followed by in agreement with several studies that claim that EVs brainstem encephalitis (n = 58; 30.5%), encephalitis are isolated from CSF only in a minority of patients, (n = 26; 13.7%), AFP/myelitis (n = 17; 8.9%) and other whereas recovery from peripheral sites such as throat, motor disorders (n = 10; 5.3%). EV-A71-C1-variant was stool/rectum and vesicle fluid is more common [3,29]. responsible for most cases (n = 140; 73.7%) compared This finding suggests that in severe neurological with EV-A71-C2 (n = 5, 2.6%) and other EV types that disease, respiratory and stool specimens, in addition included EV-D68, E-30, CV-B3, E-5, E-7, CV-A6, E-18, to the CSF and serum samples, should be submitted CV-A10, CV-A4, CV-B1, E-20, E-6 and E-9 (n = 45, 23.7%) for EV testing. (p < 0.001). According to the 3´-VP1 sequence, most EV-A71 Discussion sequences in Spain during 2016 (217/233) showed This study presents the clinical and virological a high similarity with the German strains reported characterisation of the EV-A71 strains reported in in 2015 and were classified as subgenogroup C1. Spain in 2016. We identified EV-A71 subgenogroup The fact that according to the 3Dpol sequence our C1 as responsible for a large outbreak that affected strains also grouped together with the German strains young children throughout Spain in 2016 and the clear indicates that it is very probably the same recombinant association of this infection with severe neurological variant, although whole genome sequencing would be diseases. necessary to confirm this hypothesis. Since the outbreaks of polio-like disease in Bulgaria We found an association between infection with this and Hungary in 1975 and 1978 [15,16], the number of recombinant variant and the severity of the neurological EV-A71-associated neurological diseases has been low manifestations, consistent with the German and 6 www.eurosurveillance.org Table Clinical manifestations among enterovirus-A71 patients, Spain, 2016 (n = 233) Number of cases % p value HFMD-associated Patients infected with EV-A71-C1 variant (n = 217) Brain stem encephalitis 53 4 Meningoencephalitis 47 Severe Encephalitis 18 64.5 Not applicable 1 Neurological disorders AFP/myelitis 12 Other motor disorders 10 3 Aseptic meningitis 25 4 Mild 12.4 < 0.001 Other 2 Lower respiratory tract infection 17 7.8 < 0.001 Mucocutaneous symptoms 8 3.7 < 0.001 Fever without source 14 6.5 < 0.001 Other 11 5.0 < 0.001 Patients infected with EV-A71-C1 or C2 (n = 16) Brain stem encephalitis 2 Severe 31.2 Not applicable Neurological disorders Meningoencephalitis 3 Mild Aseptic meningitis 5 31.2 0.069 1 Lower respiratory tract infection 3 18.8 0.195 Fever without source 3 18.8 0.195 AFP: acute flaccid paralysis; EV: enterovirus; HFMD: hand, foot and mouth disease. Significant variations between groups were evaluated by chi-squared test; p values comparing the number of cases with severe neurological disorders with the remaining clinical manifestations are shown. French reports [18-20]. In addition, most patients did specific area may facilitate the emergence of a strain not present HFMD before developing neurological that causes an outbreak, especially in young children manifestations (Table), also in agreement with the [40]. However, as non-polio EV infection is not a German and French reports but not with the numerous notifiable disease in Spain and asymptomatic strains publications from outbreaks in Asia-Pacific in recent may have been circulating during recent years, the real years [31-34]. Several clinical features such as young age number of EV-A71 infections could be underestimated. or fever have been associated with severe neurological Unfortunately, no seroprevalence studies are available EV-A71 disease [3]. On the other hand, the absence in Spain to confirm this hypothesis. It is important of mucocutaneous ulcers that we saw in patients in to highlight that we have identified sporadic cases our study has not been related to the development associated with this EV-A71-C1 variant in 2017 and of complicated or fatal cases by other studies [3]. 2018 (data not shown), indicating that this variant is Interestingly, most of the 15 EV-A71-C2 cases and the still circulating in Spain, but with lower incidence. The one EV-A71-C1 case unrelated to the outbreak were not seasonality of the cases during 2016, including C1 and associated with severe neurological disease. Since no C2 subgenogroups, was similar to previously described particular genogroup has been conclusively associated epidemics of EV in Spain [22,23,30]. Moreover, the with greater virulence or certain clinical manifestations geographical distribution of the outbreak indicates [5,35] and taking into account that in most cases, host that it was not restricted to the region of Catalonia, but factors play a determining role in the development of distributed throughout Spain. the disease, further experiments would need to be performed in order to describe the viral determinants As more severe neurological cases occurred during this responsible for this new recombinant strain being outbreak, there was concern among the population, associated with severe neurological pathology. the media and health professionals. Because of this concern, there was constant communication The reason why the same recombinant variant caused among microbiologists, epidemiologists and clinical such a large outbreak in Spain, while only a limited paediatricians at national level, in which the National number of cases were reported in Germany and Centre for Microbiology was involved. For this reason, France, remains unknown. Large epidemics have been we strongly believe that we studied a large and associated with genogroup replacement [5,36,37]. representative number of cases although we have the This would be in agreement with the low circulation limitation of not being able to know with certainty the of subgenogroup C1 in Spain before 2016 [22,23,30] final number of cases of the outbreak. In addition, it in contrast to the situation in Germany and France should be noted that we were informed that most severe [2,17,38,39]. Low seroprevalence against EV-A71 in a cases were diagnosed virologically, and only some www.eurosurveillance.org 7 We wish to thank I Bustillo, H del Pozo and P Higueras for mild cases were missed [41]. On the other hand, since their technical assistance. We also sincerely wish to thank the Spanish surveillance system is voluntary, there is a all technical staff from microbiology departments and medi- risk of having a bias because hospitals are more likely cal staff from paediatrics departments from all participating to send samples of severe patients. As mentioned hospitals. Some of the samples are included in an ongoing above, 777 EV-positive samples were genotyped at the project (PI15CIII-00020) which was supported by a grant by SNLRE during 2016. Of those, only 190 (25%) belonged the Health Research System (AES). to patients with severe neurological disorders. The fact that most samples received in the laboratory during Conflict of interest 2016 were from patients with mild symptoms, as well as in previous years, minimises this risk. None declared. Another limitation of our study could be that hospitals may have a bias towards reporting cases in children. Authors’ contributions Since EV infections are clearly associated with chil- Ma Montserrat Ruiz-García, Maria José González-Abad, dren, it seems that this is a bias than cannot be pre- Mercedes Alonso, Gregoria Megías, Nuria Rabella, Margarita vented; however, samples from patients of all ages del Cuerpo, Mónica Gozalo-Margüello, Alejandro González- were received in 2016. Our laboratory has previously Praetorius, Ana Martínez-Sapiña, María José Goyanes-Galán, María Pilar Romero, Andrés Antón, Manuel Imaz, Maitane described the association of some serotypes with dif- Aranzamendi, Antonio Moreno-Docón, Sonia Rey Cao and ferent age groups [23]. Our results suggest an asso- Ana Navascués: contribution of samples and clinical data, ciation of infection with EV-A71-C1 variant in young performance of the experiments. Didac Casas-Alba and children. Águeda Hernández-Rodríguez: contribution of samples and clinical data and revising the article. Cristian Launes, Carmen Muñoz-Almagro and Cristina Calvo: study concept Finally, more Spanish regions could have been affected and design, contribution of samples and clinical data and for which we may not have had data, either because revising the article. Almudena Otero: performance of the some hospitals did not send samples or because some experiments. Rubén González-Sanz and María Cabrerizo: samples received from reference hospitals, mostly in study concept and design, performance of the experiments, Barcelona and Madrid, belonged to patients admitted analysis of the data and preparation of manuscript. from the surrounding regions. 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