microorganisms Article Epidemiology of Echovirus 30 Infections Detected in a University Hospital in Catalonia, Spain, in 1995–2020 Margarita del Cuerpo 1,2, * , Jon Gonzalez de Audicana 3 , Maria Dolores Fernandez-Garcia 3 , Pilar Marín 1 , Montserrat Esteban 1 , Montserrat Español 1 , María Cabrerizo 3,4 and Núria Rabella 1,2 1 Microbiology Department, Hospital de la Santa Creu i Sant Pau, Sant Pau Biomedical Research Institute (IIB Sant Pau), 08041 Barcelona, Spain; pmarin@santpau.cat (P.M.); mesteban@santpau.cat (M.E.); mespanol@santpau.cat (M.E.); nrabella@santpau.cat (N.R.) 2 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 3 Enterovirus and Viral Gastroenteritis Unit, National Centre for Microbiology, Instituto de Salud Carlos III, 28222 Madrid, Spain; jonma_audikana@hotmail.com (J.G.d.A.); mdfernandez@isciii.es (M.D.F.-G.); mcabrerizo@isciii.es (M.C.) 4 Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: mcuerpo@santpau.cat Abstract: There is a growing interest in echovirus 30 (E30), an enterovirus responsible for neuro- logical disease and hospitalization. There are multiple studies of outbreaks, but few that study the epidemiology over long periods of time. Our study aims to describe the clinical, epidemiological and microbiological characteristics of a series of E30 infections detected over 26 years. Data were retrospectively collected from a database of all enterovirus infections identified in our laboratory.  They were detected by viral isolation or nucleic acid detection in patients presenting with respiratory  or neurological infections, rash, sepsis-like syndrome, or gastroenteritis. Enterovirus genotyping Citation: del Cuerpo, M.; Gonzalez de Audicana, J.; Fernandez-Garcia, was performed by amplification of the VP1 gene using RT-nested PCR, followed by sequencing and M.D.; Marín, P.; Esteban, M.; Español, BLAST analysis. Of the 2402 enterovirus infections detected, 1619 were linked to at least one genotype M.; Cabrerizo, M.; Rabella, N. and 173 were caused by E30. Clinical information was available for 158 (91.3%) patients. E30 was Epidemiology of Echovirus 30 associated with neurological infection in 107 (67.8%) cases and it was detected almost every year. Infections Detected in a University Phylogenetic analysis was performed with 67 sequences. We observed that E30 strains circulating in Hospital in Catalonia, Spain, in Catalonia from 1996 to 2016 belong to two lineages (E and F), although the majority cluster was in F. 1995–2020. Microorganisms 2022, 10, In 2018, lineage I emerged as the dominant lineage. 592. https://doi.org/10.3390/ microorganisms10030592 Keywords: enteroviruses; echovirus 30; central nervous system; cerebrospinal fluid; phylogenetic Academic Editor: Mario Clerici analysis; epidemiology Received: 17 February 2022 Accepted: 7 March 2022 Published: 9 March 2022 1. Introduction Publisher’s Note: MDPI stays neutral Enteroviruses (EVs) belong to the Picornaviridae family, which currently consists of 68 with regard to jurisdictional claims in genera and 158 species. The genus Enterovirus consists of 15 species, of which 4 EV species published maps and institutional affil- (A, B, C, D), with more than 100 different genotypes, and 3 rhinovirus species (A, B, C) can iations. infect humans. They are small (24–30 nm) non-enveloped viruses, consisting of a single strand of positive polarity RNA acting directly as messenger RNA, and an icosahedral capsid consisting of four proteins (VP1, VP2, VP3, VP4) [1]. Genetic differences in the VP1 Copyright: © 2022 by the authors. polyprotein give rise to the different genotypes [2,3]. Mutations and recombination events Licensee MDPI, Basel, Switzerland. are frequent in EVs. Exchanges can occur within the same genotype or between different This article is an open access article genotypes, contributing to the creation of variants and the evolution of EVs [4,5]. distributed under the terms and EVs are among the most common viruses infecting humans and are distributed world- conditions of the Creative Commons wide. Some genotypes are endemic with minor variations in circulation from year to year Attribution (CC BY) license (https:// and others are epidemic in nature, causing outbreaks in certain years. They are transmitted creativecommons.org/licenses/by/ predominantly by the fecal–oral route but also by respiratory aerosols and contact with 4.0/). vesicular fluid and surfaces contaminated with the virus. Microorganisms 2022, 10, 592. https://doi.org/10.3390/microorganisms10030592 https://www.mdpi.com/journal/microorganisms Microorganisms 2022, 10, 592 2 of 12 Up to 90% of EV infections are asymptomatic, but they can cause a wide spectrum of diseases, ranging from a mild non-specific febrile syndrome, colds, and muco-cutaneous rashes, such as herpangina and hand-foot-mouth disease (HFMD), to severe illnesses, such as pneumonia, myocarditis, pericarditis, aseptic meningitis, encephalitis or acute flaccid paralysis (AFP), which may even lead to death. They are also involved in sepsis and congenital and neonatal infections. Worldwide, EV infections account for hundreds of thousands of hospital admissions, with aseptic meningitis being the most common, and although they are a major cause of morbidity and mortality, especially in young children, notification of these cases is not compulsory in many countries, and therefore EV infection is likely to be underestimated [6–8]. No disease, clinical presentation or symptom can be attributed to a single genotype. However, different genotypes have been associated with certain clinical pictures, such as po- lioviruses (PVs) and poliomyelitis [9], enterovirus D68 (EV-D68) and severe respiratory and neurological presentations [10], enterovirus A71 (EV-A71) and HFMD and encephalitis [11], or echovirus 30 (E30), long described as a cause of aseptic meningitis worldwide [12–16]. E30 is an EV genotype frequently detected in our environment [17]; it belongs to the EV B species, is antigenically heterogeneous, and is classified phylogenetically into nine lineages [18,19]. E30 outbreaks show a cyclical incidence pattern of 3 to 5 years that is generally associated with the rapid spread of different strains [20–22]. Many studies describe outbreaks of aseptic meningitis caused by E30, but there are few about the epidemiology and molecular evolution of this genotype over a long period. Given the growing interest in E30 as a cause of neurological disease and hospitalization [23], our study aims to describe the clinical, epidemiological and microbiological characteristics of a series of E30 infections detected in our laboratory over a period of 26 years. 2. Materials and Methods 2.1. Study Design Epidemiological, microbiological, and clinical data were retrospectively collected from all E30 infections identified in the virology laboratory of a university hospital (Hospital Santa Creu i Sant Pau, Barcelona) from 1995 to 2020. This hospital is a 548-bed reference medical center for highly complex pathologies that provide healthcare services for a popula- tion of 407,550. The laboratory is accredited as a sub-national polio laboratory by the World Health Organization (WHO) and has participated in a Spanish EV molecular surveillance network since 1998. 2.2. Clinical Samples For routine diagnosis of EV/PV infections, the laboratory received annually a mean of 2180 clinical samples from patients, mainly pediatric, with the following diseases: respiratory, neurological, muco-cutaneous, sepsis-like or gastroenteric. The main types of biological samples were respiratory (nasopharyngeal aspirates, bronchoalveolar lavages, pharyngeal exudates), stools, cerebrospinal fluids (CSF) and skin exudates. 2.3. Methods 2.3.1. EV Isolation Until 2007, the only EV detection method used by the laboratory was the culture of clinical samples on different cell lines: human fetal lung MRC-5 (RD BIOTECH (Be- sançon, France), human lung carcinoma A549 (Vircell S.L. (Granada, Spain), human epi- dermoid laryngeal carcinoma HEp2 (Vircell S.L. (Granada, Spain) and human embryonal rhabdomyosarcoma RD (Enterovirus and Viral Gastroenteritis Unit. National Centre for Microbiology. Instituto de Salud Carlos III, Madrid, Spain). Samples were processed and inoculated following the WHO polio laboratory manual for PV/EV isolation [24,25]. Viral cultures were incubated at 37 ◦ C and observed daily under inverted microscopy for 14 days to detect a cytopathic effect compatible with EV infection. An indirect immunofluorescence technique with a mixture of EV-specific monoclonal antibodies (Enterovirus Screening Set Microorganisms 2022, 10, 592 3 of 12 LIGHT DIAGNOSTICS™ and D3 IFA Enterovirus Identification Kit Diagnostic HYBRIDS) was used for confirmation of the EV infection. 2.3.2. EV Detection A commercial reverse transcription-polymerase chain reaction (RT-PCR) assay (XpertEV, Cepheid, Sunnyvale, CA, USA), designed to detect EV RNA directly in clinical samples in the 50 UTR untranslated region of the genome, was adopted in 2007. 2.3.3. EV Genotyping EV-positive samples and isolates were sent to the Enterovirus Unit of the National Centre for Microbiology (Instituto de Salud Carlos III, Madrid, Spain) for EV type char- acterization. EV genotyping was performed by amplification of the 30 -part of the VP1 gene using conventional RT-nested PCRs specific for EV-A, B, C or D, as previously re- ported [26,27], followed by sequencing and BLAST analysis of the sequences obtained (http://blast.ncbi.nlm.nih.gov/). Specific type was assigned when the homology in the nucleotide sequence was higher than 75%. 2.3.4. Phylogenetic Analysis A phylogenetic analysis was carried out with 67 E30 available sequences detected between 1996 and 2018, the prototype strains (Bastianni, Frater) and 50 sequences in the same 30 -VP1 region (420 bps) from other countries available in GenBank. Alignments were performed with ClustalW [28] and the phylogenetic tree was built with the neighbor-joining method, the maximum composite likelihood distance model, and 1000 pseudo-repeats, such as the bootstrap method, in the MEGA X program [29]. All VP1 sequences were deposited in GenBank. Genome sequences obtained in this study were deposited in GenBank (accession numbers OM925759-OM925825). 2.3.5. Ethics Statement The clinical and epidemiological data were treated anonymously and their study was approved by the clinical research ethics committee of the Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau—IIB Sant Pau with protocol code IIBSP-ENT-2019-42. 3. Results 3.1. EV Detection and Genotyping During the 26-year study period, 2402 patients were diagnosed with EV infection. A total of 2929 EV-positive samples were included in this study, and 2033 (69.4%) of these were genotyped. The specific EV type was determined in 1619 patients. Four predominant genotypes were found: echovirus 11 (E11) in 199 cases (12.3%), echovirus 6 (E6) in 186 cases (11.5%), coxsackievirus B5 (CVB5) in 124 cases (7.6%) and E30 in 173 cases, representing 10.7% of the total genotyped EV. 3.2. Demographic, Epidemiological and Clinical Characteristics of E30 Infections The male/female ratio of the E30-positive patients was 1.5:1 (106/67) with a mean age of 8 years, ranging from 7 days to 81 years. Of the total E30 infections, 87 (50.4%) were in children between 0 and 3 years, with infants up to 3 months of age accounting for 16% of the total patients. Clinical information from 158 (91.3%) patients was available. E30 infections associated with the central nervous system (CNS) were 107/158 (67.8%), with most of them (103, 96.3%) with symptoms suggestive of aseptic meningitis, while only 4 cases were diagnosed with meningoencephalitis or encephalitis. Non-specific febrile syndrome (or without further information) was found in 34/158 (21.5%) cases, respiratory diseases in 12/158 (7.6%) and muco-cutaneous lesions in 5/158 (3.1%) cases (Table 1). Microorganisms 2022, 10, 592 4 of 12 Table 1. Demographic and clinical characteristics of 173 patients with E30 infection. Age Group E30 Infections Percentage (%) 0–3 months 28 16.2 4–23 months 25 14.5 2–3 years 34 19.7 4–5 years 24 13.9 6–10 years 28 16.2 11–17 years 10 5.8 18–40 years 16 9.2 >40 years 6 3.4 not specified 2 1.1 Sex Female 67 38.7 Male 106 61.3 Patients with clinical information 158 91.3 Non-specific febrile syndrome 34 21.5 Exanthema 5 3.1 Respiratory symptoms 12 7.6 Neurological symptoms 107 67.8 Meningitis 103 96.2 Meningoencephalitis 2 1.9 Encephalitis 2 1.9 A total of 254 samples from these 173 patients were studied. Two hundred and thirty-two samples were E30 positive, of which 87 (37.5%) were CSF, 99 (42.7%) were respiratory (mainly nasopharyngeal aspirate mucus and/or pharyngeal exudates and one bronchoalveolar lavage), 42 (18.1%) were fecal, 2 were urine and there were 2 skin exudates (0.9% each) (Table 2). Table 2. Types of samples studied in 173 cases of E30 infections. Sample n E30-Positive Percentage (%) CSF * 97 87 89.7 Respiratory sample 108 99 91.7 Feces 42 42 100 Urine 5 2 40 Skin exudates 2 2 100 Total 254 232 91.3 Cases with multiple specimens analyzed (n = 48) Cases Percentage (%) CSF + RS ** + feces + urine 1 0.6 CSF + RS + urine 2 1.1 CSF + RS + feces 22 12.7 CSF + RS 10 5.8 CSF + feces 3 1.7 RS + feces 8 4.6 RS + feces + urine 1 0.6 RS + urine 1 0.6 * CSF, cerebrospinal fluid; ** RS, respiratory sample. In 48 patients (27.8%) multiple samples were studied, with CSF, respiratory and stool samples being the most frequent combination (22 patients) (Table 2). In the remaining 125 patients (72.2%) only one type of sample was studied. In 38 patients with CNS involve- ment, more than one type of specimen was studied. In these cases, the detection of E30 in Microorganisms 2022, 10, x FOR PEER REVIEW 5 of 12 Microorganisms 2022, 10, 592 5 of 12 Cases with multiple specimens analyzed (n = 48) Cases Percentage (%) CSF was efficient in 76% CSF + RS ** + feces + urine (29/38) of1the cases, in respiratory 0.6 specimens in 80% (27/34) and in feces in 100% CSF + RS + urine (26/26). 2 1.1 CSF +In RSaddition + feces to E30 infection, other 22 viruses were12.7 detected in 10 (5.8%) patients, in the same or CSF + RS a different sample. In 6 cases, 10 other EV types were 5.8 identified: in one patient E6 was co-detected CSF + feces in CSF; in another, E303was found in CSF and 1.7 feces and E11 in the respiratory sample; RS + feces in two cases, E30 and EV-A71 8 were co-detected 4.6 in feces; one E30-positive patient in RS feces + feceshad an E11 in the respiratory + urine 1 sample; finally, 0.6 in one patient, E30 was detected in RS urine + urineand E11 in the respiratory 1 sample. In addition, 0.6 other respiratory viruses (two adenoviruses-ADV-, * CSF, cerebrospinal fluid;one**parainfluenza RS, respiratory3sample. virus -PIV3-, and one respiratory syncytial virus -RSV-) were found in four cases. E30 was detected in almost all the years recorded, with an increase in incidence every 2–5 2-5 years, which which did didnot notalways alwayscoincide coincidewith withthe years the of of years highest observation highest of overall observation EV of overall infections. The The EV infections. yearyear withwith the highest number the highest of E30 number of detections in this E30 detections instudy was 2007, this study with was 2007, other upsurges with other in 1996, upsurges 2000-01, in 1996, 2005, 2005, 2000-01, 2012-13, 2015-16 2012-13, and 2018. 2015-16 Outbreaks and 2018. sometimes Outbreaks some- occurred in a single times occurred in a year singleoryear wereorextended over a 2-year were extended over aperiod 2-year (Figure 1, Table 1, period (Figure 3).Table Even 3). in the Even years with in the the with years highest thedetection of E30, this highest detection of did E30,not usually this did notexceed 10–35% usually exceed of10–35% all the EV of infections all the EV detected, infectionsexcept in 2007 detected, when except in E30 2007cases whenmade up more E30 cases made than up50% moreof than the total 50%EVof detected. the total EV detected. Figure 1. Annual distribution of E30 detections vs. other EV genotypes. Figure 1. Annual distribution of E30 detections vs. other EV genotypes. TableRegarding 3. E30 infections with respect the seasonal to the total distribution of of E30EVinfection, genotypedalthough by year. the virus was detected in practically EV Year all Detection months of the year, there were Genotyped EV two (%) periods of E30increased Detectiondetection: (%) one between 1995 May and 23 July, which accounted 19 (82.6)for almost half of all cases, 0 and another shorter period 1996 in autumn 42 (November and December). 19 (45.2) Comparing the 7number (36.8) of cases associated with 1997 CNS infection 76 with the rest of the EV 35 (46) infections, we find that in the months of June and 3 (8.6) July, 1998 the number76 of patients with neurological 28 (36.8) involvement is three 0 times higher than in the rest 1999of the year, 115while in the autumn 52peak (45.2)(November, December), 0 that number increases in parallel with 2000 111the other infections,62 and (55.9)in the winter months 13 (January, (21) February), it is surpassed 2001 by the 93 other infections (Figure 2). 70 (75.3) 9 (12.9) 2002 98 68 (69.4) 4 (5.9) 2003 96 85 (88.5) 0 2004 74 54 (73) 1 (1.8) 2005 151 119 (78.8) 17 (14.3) 2006 132 94 (71.2) 2 (2.1) Microorganisms 2022, 10, 592 6 of 12 Microorganisms 2022, 10, x FOR PEERTable REVIEW 3. E30 infections with respect to the total of EV genotyped by year. 6 of 12 Year EV Detection Genotyped EV (%) E30 Detection (%) 1995 2007 137 23 19 (82.6) 91 (66.4) 0 48 (52.7) 1996 2008 129 42 19 (45.2) 78 (60.5) 7 (36.8) 1 (1.3) 1997 76 35 (46) 3 (8.6) 2009 119 92 (77.3) 3 (3.3) 1998 76 28 (36.8) 0 2010 1999 162 115 116 (71.6) 52 (45.2) 7 (6) 0 2011 2000 122 111 74 (60.6) 62 (55.9) 1 (1.4) 13 (21) 2012 2001 130 93 67 (51.5) 70 (75.3) 7 (10.4) 9 (12.9) 2013 2002 63 98 43 (68.2) 68 (69.4) 15 (34.9) 4 (5.9) 2003 2014 64 96 85 (88.5) 42 (65.6) 0 0 2004 2015 70 74 54 (73) 52 (74.3) 1 (1.8) 8 (15.4) 2005 2016 91 151 119 67 (73.6) (78.8) 17 (14.3) 11 (16.4) 2006 2017 92 132 94 71 (77.2)(71.2) 2 (2.1) 2 (2.8) 2007 137 91 (66.4) 48 (52.7) 2018 72 61 (84.7) 13 (21.3) 2008 129 78 (60.5) 1 (1.3) 2019 48 47 (98) 1 (2.1) 2009 119 92 (77.3) 3 (3.3) 2020 2010 16 162 15 (93.7) 116 (71.6) 0 7 (6) Total 2011 2402 122 1619 74 (67.4) (60.6) 1731(10.7) (1.4) 2012 130 67 (51.5) 7 (10.4) 2013Regarding the63 seasonal distribution43of(68.2) E30 infection, although15the (34.9) virus was detected 2014 64 42 (65.6) 0 in practically all months of the year, there were two periods of increased detection: one 2015 70 52 (74.3) 8 (15.4) between May and July, which accounted for almost half of all cases, and another shorter 2016 91 67 (73.6) 11 (16.4) period 2017 in autumn (November 92 and December). 71 (77.2)Comparing the number 2 (2.8) of cases associated with 2018 CNS infection 72 with the rest of the EV infections, 61 (84.7) we find that in the months of June 13 (21.3) and 2019July, the number 48 of patients with neurological 47 (98) involvement is1 three (2.1) times higher than in2020 16 the rest of the year, 15 (93.7) while in the autumn 0 peak (November, December), that number Total 2402 1619 (67.4) increases in parallel with the other infections, and in the winter months173 (10.7)(January, Febru- ary), it is surpassed by the other infections (Figure 2). (a) (b) Figure 2. Figure 2. (a) (a) Seasonal Seasonaldistribution distributionofofall allE30 E30infections infections included included in in this this study; study; (b)(b) seasonal seasonal distribu- distribution tion of E30-positive cases with central nervous system (CNS) diseases versus other clinical presen- of E30-positive cases with central nervous system (CNS) diseases versus other clinical presentations. tations. 3.3. E30 Phylogenetic Analysis 3.3. E30 Phylogenetic Analysis To investigate the spatiotemporal relationships among E30 strains detected in our To from hospital investigate 1995 tothe spatiotemporal 2020, relationships 67 available VP1 among sequences were E30 strains compared detected to other in our homologous hospital from 1995 to 2020, 67 available VP1 sequences were compared to other strains from different countries worldwide. According to the phylogenetic classification in homolo- 9gous strains lineages frompreviously (A–I), different countries publishedworldwide. According [18,20] sequences fromto the to 1996 phylogenetic 2006 belonged classifi- to E cation and in 9 most F, and lineages (A–I), of the E30 previously published detected between 2007[18,20] sequences and 2016 from 1996 were grouped toin in F, 2006 be- which longed tosubclades different E and F, andcan most of the E30 detected be distinguished. Finally,between all VP1 2007 and 2016 sequences fromwere 2018grouped in clustered F, in which within different lineage I, with subclades can be distinguished. a higher nucleotide Finally, divergence from C–Hall VP1 sequences (26.2–35.5%). from 2018 No sequences clustered from withinbelonged Catalonia lineage I,towith A–D a higher and G–Hnucleotide lineages divergence (Figure 3). from C-H (26.2–35.5%). No sequences from Catalonia belonged to A-D and G-H lineages (Figure 3). Microorganisms 2022, 10, Microorganisms2022, 10, x592 FOR PEER REVIEW 7 of 12 7 of 12 Figure 3. Phylogenetic tree of the 30 -VP1 region with E30 sequences from 1996–2020 classified into 9 lineages, A–I. The tree was reconstructed using the neighbor-joining method and the maximum composite likelihood model. The tree was rooted with the prototype sequences Bastianni (AF311938) and Frater (AF081341). Bootstrap resampling (1000 replicates) was used to determine grouping robustness. Values of >70% are shown. E30 sequences obtained in this study are colored according to the year of detection (yellow 1996–1997; green 2001–2006; red 2007–2009; violet 2010–2013; blue 2015–2018). Microorganisms 2022, 10, 592 8 of 12 4. Discussion This study presents the epidemiology and molecular evolution of the E30 detected in a university hospital in Barcelona (Catalonia, Spain) over a period of 26 years. E30 has been widely detected for decades and is described as one of the most predomi- nant EV genotypes in multiple reports [20,30–32]. In our study, it is the third most detected EV type, after E6 and E11, and was found in approximately 11% of cases. However, our percentage is slightly lower compared to data for the entire Spanish territory [17,33], where E30 is the most prevalent genotype and is reported to exceed 25%. We found that in almost 68% of cases with E30 detection, neurological involvement was recorded, almost entirely meningitis. This result was to be expected, as E30 is one of the genotypes most frequently associated with aseptic meningitis [34,35], with multiple outbreaks widely documented throughout the world. However, we also detected E30 related to other clinical manifestations, such as non-specific fever and respiratory symptoms. Milder illnesses, such as uncomplicated respiratory infections, may be underestimated because clinical samples from these pathologies are not routinely collected for testing. As is the case for infections with other EV genotypes [36], E30 was detected mainly in children: 50% of the patients were under three years old, with a high incidence (16% of the total) in babies from 0 to 3 months. This could be because babies and very young children are more prone to infection as they have not yet acquired immunity, and medical attention is sought quickly for symptoms, such as fever or irritability in this population. Some studies with patients of all ages report that in addition to the pediatric age group, young adults also have a high incidence of infection [20,37] but this was not observed in our series. We found that in our region, E30 can be considered an endemic EV since it circulates almost every year (without detection only in 6 out of 26 years), with an increase in cases every 2–5 years in which the number of E30 infections detected ranges from 15% to 54% of the total EVs, with an outbreak duration of 1 to 2 years, in accordance with other studies. It is possible that after periods of intense activity, in which population immunity is created for circulating variants, detections drop until different variants break out, causing a significant increase in cases to reappear [20–22]. In our study, the emergence of the new F and I lineages correlates with an increase in the incidence in Catalonia in the years 2005–2007 and 2018, respectively. EV infections present seasonality in temperate zones, being more frequent in sum- mer and autumn, and a homogeneous distribution throughout the year in tropical and subtropical zones [38]. In our series, E30 detections follow a seasonal pattern, with a considerable increase in cases in late spring and early summer, but we also found a small increase in autumn (November and December). If we separate the cases with neurological symptoms, we observe a large peak in June and July, in accordance with previous reports of aseptic meningitis outbreaks [13]. However, if we look only at non-neurological cases, the seasonality is different, with three peaks in February, May, and November-December. It is possible that this increase is due to the fact that in the autumn and winter months, respiratory infection is more frequent than neurological infection. However, more studies with a greater number of cases of respiratory infection due to E30 would be needed to confirm this hypothesis. In almost 6% of E30 cases, other viruses were co-detected, which was favored by the study of multiple samples per case. The most frequent association was between E30 and other EV types (E6, E11 and EV-A71), and respiratory samples were found to have the greatest viral diversity. The versatility of the technique used to detect EV in respiratory samples, cell culture in various cell lines, allowed other conventional respiratory viruses (ADV, RSV, PIV3) to be detected in 4 cases. It should be noted, that in one case we found two EVs (E30 and E6) in the CSF specimen of an elderly man. Coinfections with more than one virus are widely described in the literature [39–42]. Some authors suggest that simultaneous infection by more than one virus generates more severe symptoms [43] and that infection by more than one genotype can generate recombination events between different species [4,44], Microorganisms 2022, 10, 592 9 of 12 giving rise to new variants. In our series, the number of co-detections is too small to draw conclusions, and it would be of interest to conduct more studies with a larger number of cases to investigate the clinical importance of coinfection. Given that the finding of the virus in CSF samples confirms the neurological infection, it is the most studied sample type in the numerous studies on outbreaks of meningitis caused by E30. We found that CSF is a good sample to detect E30 in aseptic meningitis (90% of the CSF samples studied were positive) compared to other genotypes found in cases of neurological involvement, such as EV-A71 [11,45,46] or EV-D68 [27] where they are barely detected. However, these last two genotypes are more associated with encephalitis or AFP than with meningitis [35], which could be the reason why the virus was not found in the CSF at the time of the study [47]. It should be noted that in our series, 10% of the CSF samples were negative, and the infection was diagnosed by the analysis of other clinical samples, such as nasopharyngeal mucus and/or feces, where the efficiency of nasopharyngeal exudate or feces was 80% and 100%, respectively. It is of interest to study samples other than CSF in cases where neurological symptoms are present. The resulting phylogenetic tree showed a clear turnover of E30 lineages over the sampled years, in accordance with other studies [18–20]. A recent study analyzing the evolution of E30 strains in Spain observed a genetic turnover in which an emerging lineage I replaced previous lineages circulating in Spain. Moreover, this study observed that lineage F contained most of the E30 strains previously identified in Spain from 1996 to 2018 [19]. In agreement with these observations, the majority of strains in the present study (from 1997 to 2016) clustered in lineage F. Several sub-lineages could be distinguished in this lineage, which correlated with an increase in E30 incidence in 2007, 2013 and 2015-16. We similarly note that all sequences from 2018 belong to the emerging lineage I. According to Benschop et al. [20], the E30 upsurge observed in several European countries during 2018 was caused by the appearance of two different clades, G1 and G6 (corresponding to lineages E and I, respectively). In this study, two analyzed sequences obtained from Catalonia in 2018 (MK815445 and MZ389230) clustered in these two respective clades. It is noteworthy, that in our study all sequences from 2018 belonged only to lineage I, a finding in accordance with Gambaro et al. [19], who reported that E30 strains from 2018, sampled in Andalusia (South Spain), all belonged to lineage I. Although these phylogenetic analyses show the emergence of lineage I in Spain, overall, E30 was not the most frequent EV genotype detected that year, being third in predominance after EV-D68 and E9, respectively, and in 2019, it accounted for 14.5% of total typed EV (data not published). It is possible that the present study suffered from a selection bias as the majority of individuals diagnosed with EV infection were patients who attended our hospital with clinical symptoms significant enough to warrant a hospital consultation and for physicians to collect clinical samples for laboratory analysis. 5. Conclusions In the region of Catalonia, E30 is an endemic EV that circulates almost every year with a peak of cases in late spring and early summer. E30 was detected from CSF specimens of most of the patients with neurological involvement, but not in all. The phylogeny showed that E30 strains circulating in Catalonia shifted from lineages E and F to lineage I in 2018. The emergence and dominance of a single lineage in 2018, replacing previous dominant lineages, highlights the importance of continuous surveillance of E30 circulation as well as other clinically relevant EVs. Author Contributions: Conceptualization, M.d.C. and N.R.; methodology, M.d.C., J.G.d.A., M.D.F.- G., P.M., M.E. (Montserrat Esteban), M.E. (Montserrat Español), M.C. and N.R.; formal analysis, M.d.C., J.G.d.A. and M.D.F.-G.; writing—review and editing, M.d.C., M.C. and N.R.; supervision, M.C. and N.R. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Microorganisms 2022, 10, 592 10 of 12 Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and as mentioned before this study was approved on 25 June 2019 by the clinical research ethics committee of the Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau—IIB Sant Pau with protocol code IIBSP-ENT-2019-42. Informed Consent Statement: The clinical research ethics committee of the Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau—IIB Sant Pau waived the requirement of patient consent. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. Internationall Committee on Taxonomy of Viruses ICTV Picornaviridae. Available online: https://talk.ictvonline.org/ictv- reports/ictv_online_report/positive-sense-rna-viruses/w/picornaviridae (accessed on 15 November 2021). 2. Palacios, G.; Casas, I.; Tenorio, A.; Freire, C. 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