Infectious Diseases, 2015; Early Online: 1–5
BRIEF REPORT
Coexistence of two clades of enterovirus D68 in pediatric Swedish patients in the summer and fall of 2014
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ROBERT DYRDAK1,2, MARIA ROTZÉN-ÖSTLUND1,2, AGNETA SAMUELSON1, MARGARETA ERIKSSON3 & JAN ALBERT1,2 From the 1Department of Clinical Microbiology, Karolinska University Hospital, 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, and 3Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden
Abstract In 2014, an outbreak of enterovirus D68 (EV-D68) was observed in North America, with cases of severe respiratory illness and a possible etiological link to cases of acute flaccid paralysis. EV-D68 has also been reported from European countries, but no data from Sweden are available. This study investigated respiratory specimens collected during July–October 2014 from 30 Swedish children aged 0–9 years who were positive for enterovirus and/or rhinovirus in routine clinical PCR. Seven samples were typed as EV-D68 by VP4/VP2 sequencing. Two genetically distinct EV-D68 variants coexisted. Six viruses belonged to clade B, the variant involved in the North American outbreak, and one virus belonged to clade A. Respiratory illness was the major symptom among EV-D68 infected patients and all fully recovered. This is the first report of EV-D68 in Sweden. Considering the current epidemiological situation, genotyping and specific EV-D68 testing should be considered in patients with severe respiratory illness who test positive for enterovirus or rhinovirus in routine diagnostics.
Keywords: Enterovirus D68, pediatric, respiratory infection, VP4/VP2 sequencing
Introduction Enterovirus D68 (EV-D68) is a small, non-enveloped, single-stranded RNA virus classified within the enterovirus D species of the Picornaviridae family. Unlike other enteroviruses, but similar to rhinoviruses, EV-D68 is acid-fragile, grows poorly at 37°C, and has primarily been associated with respiratory symptoms [1]. EV-D68 has until recent years rarely been reported. In the National Enterovirus Surveillance System in the USA there were only 26 reports of EV-D68 between 1970 and 2005 [2]. In all cases but one, the virus was detected in respiratory specimens [2]. However, a study on serum samples from pregnant women in Finland collected in 1983, 1993, and 2002 showed neutralizing antibodies against the Fermon/prototype EV-D68 strain in 100% of the subjects, indicating that exposure to EV-D68 had been common [3]. In a study of Finnish military conscripts with acute respiratory infection in 2004–2005,
EV-68 was found in 3 of 32 sputum specimens [4]. Retrospective analyses have shown circulation of EV-D68 in the Netherlands during 2011–2014, and Cambodia in 2009–2012, where it was the most common virus in adults with unexplained respiratory illness [5,6]. In contrast, no EV-D68 was detected in a study of 456 respiratory samples collected in Edinburgh, Scotland in 2006 and 2007 [7]. A change in the epidemiological pattern of EV-D68 infections has occurred in 2014, with the United States experiencing a nationwide outbreak with more than 1000 cases of severe respiratory illness primarily affecting children [8,9]. In addition, EV-D68 has been detected in cases of acute flaccid paralysis, but it is still uncertain if the virus caused this condition [10–12]. Increased rates of respiratory EV-D68 infections and cases with neurological symptoms have also been reported from Canada [13–15]. In Europe, information is more limited, but an increase of EV-D68 in
Correspondence: Robert Dyrdak, Department of Clinical Microbiology, L2:02, Karolinska University Hospital, SE-171 76, Sweden. Tel: ⫹ 46 8517 72593. Fax: ⫹ 46 830 8099. E-mail: [email protected] (Received 3 March 2015 ; accepted 29 April 2015) ISSN 2374-4235 print/ISSN 2374-4243 online © 2015 Informa Healthcare DOI: 10.3109/23744235.2015.1047402
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2014 has been reported from the Netherlands [5,16], Spain [17], and Norway [18], and acute flaccid paralysis associated with EV-D68 infection was reported for one patient in France and two patients in Norway [19,20]. The epidemiology of EV-D68 in Sweden was unknown and therefore the aim of this study was to investigate the possible presence of the virus in Stockholm, Sweden.
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Materials and Methods Respiratory samples positive for enterovirus and/or rhinovirus in routine clinical PCR testing at the Department of Microbiology, Karolinska University Hospital between July and October 2014 were identified. During this period, 428 respiratory samples from pediatric patients aged 0–9 years had been submitted for analysis of enterovirus and rhinovirus. These assays were done with in-house real-time PCRs using previously described methods and primers [21,22]. Enterovirus was detected in 14 samples, rhinovirus in 80 samples, and dual reactivity in 91 samples, which was not surprising because the assays are known to sometimes cross-react. From these samples we selected 30 specimens (10 positive for enterovirus, 10 for rhinovirus, and 10 positive for both viruses). Of the 30 samples, 25 were nasopharyngeal aspirates, 2 were nasopharyngeal secretions, 1 bronchoalveolar lavage, 1 tracheal secretion, and 1 sampling from the nose. The study was approved by Regional Ethical Review board in Stockholm, Sweden (registration no. 2014/2233). For enterovirus/rhinovirus genotyping RNA was extracted using the MagAttract Virus Mini M48 Kit on a BioRobot M48 (Qiagen, Sollentuna, Sweden) for samples with threshold cycle (Ct) values below 30 in the routine real-time PCR (n ⫽ 20). For samples with higher Ct values (n ⫽ 10), RNA was manually extracted using RNeasy Lipid Tissue Mini Kit (Qiagen), which in our hands is more efficient than automated RNA extraction. RNA was eluted in 100 μl (BioRobot M48) or 40 μl (manual extraction) nuclease-free water and 7 μl of RNA template was used for nested reverse transcriptase (RT)-PCR using published primers targeting the VP4/VP2 region [7]. A one-step Superscript-Platinum Taq (Life Technologies, Stockholm, Sweden) was used for the first, outer PCR and Platinum Taq for the second, nested PCR. The PCR cycling profile was: reverse transcription for 30 min at 43°C, followed by denaturation for 2 min at 94°C, and 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C, and extension for 60 s at 68°C, followed by final extension of 5 min at 68°C. The RT step was omitted in the second PCR. PCR-positive samples were purified using the Agencourt AMPure XP PCR
Purification Kit (Beckman Coulter, Bromma, Sweden), eluted with TE buffer (Invitrogen, Life Technologies, Stockholm, Sweden), and were sent for bidirectional Sanger sequencing using the inner PCR primers as sequencing primers. Sequencing was done on an ABI 3730 instrument at the KIGene sequencing facility, Karolinska Institute. The VP4/ VP2 sequences were manually aligned to reference sequences from www.picornaviridae.com using BioEdit [23]. The enterovirus species and type were determined by maximum likelihood phylogenetic trees constructed using PhyML [24] with the GTR⫹ I⫹ G substitution model. Additional EV-D68 sequences were added for detailed phylogenetic analyses. The Swedish VP4/VP2 EV-D68 sequences have been deposited in Genbank under accession numbers KP745744–KP745750. Results We investigated nasopharyngeal samples from children aged less than 10 years that had tested positive for enterovirus (n ⫽ 10), rhinovirus (n ⫽ 10) or enterovirus and rhinovirus (n ⫽ 10) in routine diagnostics at the Karolinska University Hospital between July and October 2014. Sequencing of the VP4/VP2 region was successful for 22 of 30 samples, of which RNA had been extracted manually in 4 samples and by extractor in 20 samples. Most sequencing failures occurred in samples with high Ct values in the routine real-time PCR assays, i.e. in samples with low virus content. Phylogenetic tree analysis showed that 7 of the 22 viruses were genotyped as EV-D68. These samples were collected in August and September of 2014 and were positive in the routine enterovirus PCR. Three samples also cross-reacted in the routine rhinovirus PCR. Of the remaining 15 viruses, 1 was typed as echovirus 11 within the enterovirus B species and 14 viruses were typed as different human rhinoviruses (HRV-A1, HRV-A12, HRV-A15, HRVA38, HRV-A58, HRV-A63, HRV-A80, HRV-A101, HRV-B70, HRV-B86, HRV-C6, HRV-C11, HRVC18, HRV-C45). The characteristics of the seven children with EV-D68 infection are shown in Table I. All children except one had respiratory symptoms and five had underlying diseases. Six of seven children were inpatients and two children had a short stay in the intensive care unit (ICU), but all children were discharged without remaining symptoms (apart from underlying diseases in five children). In six children no other respiratory pathogen was detected. In one child bocavirus was detected, but the virus levels were low, suggesting a carrier state rather than a symptomatic infection. Children infected with EV-D68 were
Enterovirus D68 in Sweden
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Table I. Characteristics of the seven Swedish children infected with EV-D68. Patient no. 1 2 3 4 5 6 7
Age
Gender
Symptoms
Underlying disease
9.0 years 2 months 3.8 years 3.8 years 1.2 years 3.2 years 5.3 years
F M M M M F M
Respiratory symptoms Respiratory symptoms Viral syndrome Bronchitis Viral pneumonia Respiratory symptoms Respiratory symptoms
Neuromuscular disease Recurrent wheezing No No Recurrent wheezing Recurrent wheezing Asthma
Inpatient ICU days Yes Yes No Yes Yes Yes Yes
1 0 0 0 1 0 0
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F, female; ICU, intensive care unit; M, male.
significantly older (median age 3.8 years, range 2 months to 9 years) than patients infected with rhinoviruses and echovirus 11 (median age 4 months, range 1 week to 6.8 years) (p ⫽ 0.0004, Mann-Whitney U test). Among the 23 children with other enterovirus/rhinovirus infections, respiratory disease was diagnosed in 12 patients, 7 patients had underlying conditions, and 22 were hospitalized, of which 4 were treated in the ICU. Compared with patients infected with EV-D68, patients infected with other rhinoviruses or enteroviruses less frequently had
respiratory illness as the dominating symptom, but the difference was not statistically significant (p ⫽ 0.21, Fisher’s exact test). The relationship between the seven Swedish EV-D68 viruses and international EV-D68 viruses was investigated by a more detailed phylogenetic tree analysis (Figure 1). EV-D68 clade assignment was done according to Tokarz et al. [25]. The analysis showed that six of the seven viruses belonged to clade B of EV-D68 and were closely related to each other as well as to viruses collected during the 2014
Figure 1. Phylogenetic relationships of the Swedish and international EV-D68 strains constructed using an unambiguous alignment (435 base pairs) of sequences from the VP4/VP2 region of the genome and the PhyML software with the GTR⫹ I⫹ G substitution model. Sequences from Sweden have red identifiers. The tree was midpoint rooted and the scale bar indicates the number of nucleotide substitutions per site (according to the GTR⫹ I⫹ G substitution model).
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outbreaks of EV-D68 infections in the USA and other countries. The remaining virus was genetically distinct from the six other viruses and clustered within clade A of EV-D68.
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Discussion This study for the first time reports on the presence of EV-D68 in Sweden. Although the study was small, it demonstrates that infections with EV-D68 occurred among Swedish children in the late summer and early fall of 2014. Six of the seven viruses belonged to clade B of EV-D68 and were closely related to each other and to viruses from the current North American outbreak. Interestingly, one EV-D68 virus infection with a clade A virus was also documented. Thus, two genetically distinct variants of EV-D68 coexisted in Stockholm, Sweden during the study period. The coexistence of two clades of EV-D68 means that the full epidemiological picture, such as an epidemiological link to the EV-D68 outbreak in North America, cannot be obtained by simple EV-D68 diagnostics, but requires more precise genotyping. Six of the seven EV-D68-infected children in the present study were admitted to hospital and two spent 1 day in the ICU. However, all children survived and were relatively rapidly discharged from hospital without remaining symptoms. To our knowledge there have been no reports in Sweden of increased frequency of severe respiratory infections or of unexplained acute flaccid paralysis. However, genotyping is not routinely done for respiratory samples positive for enterovirus or rhinovirus in Sweden. In addition, there has been no systematic testing for respiratory infections in children with neurological symptoms. This means that infections with EV-D68 may have gone unnoticed. Children with EV-D68 infection were significantly older than children infected with rhinoviruses and other enteroviruses. The reason for this is unclear, but it is possible that pre-existing immunity was less common to EV-D68 than to the other viruses, which would allow for infection in higher age groups. Another possibility is that EV-D68 caused more severe symptoms in older children than the other viruses and that this led to more frequent hospitalization and viral diagnostics for EV-D68-infected children in higher age groups. The study was not designed or powered to allow estimation of the prevalence and incidence of EV-D68 in Sweden, but we are currently performing a larger survey to gain better insight in this question. In light of the current outbreak of EV-D68 in North America and increasing evidence of detection also in Europe, our documentation of EV-D68 in Sweden indicates that enterovirus typing should be
considered in cases of severe respiratory illness or acute flaccid paralysis to gain more insight into the epidemiology and etiology of such infections.
Acknowledgments We would like to thank Eva Eriksson from the Department of Clinical Microbiology, for expert technical assistance, and Dr Mia Brytting from the Public Health Agency of Sweden, for valuable discussions. Declaration of interest: No funding was received. No conflicting interests.
References [1] Oberste MS, Maher K, Schnurr D, Flemister MR, Lovchik JC, Peters H, et al. Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses. J Gen Virol 2004; 85:2577–84. [2] Khetsuriani N, Lamonte-Fowlkes A, Oberst S, Pallansch MA. Enterovirus surveillance – United States, 1970–2005. MMWR Surveill Summ 2006;55:1–20. [3] Smura T, Ylipaasto P, Klemola P, Kaijalainen S, Kyllonen L, Sordi V, et al. Cellular tropism of human enterovirus D species serotypes EV-94, EV-70, and EV-68 in vitro: implications for pathogenesis. J Med Virol 2010;82:1940–9. [4] Savolainen-Kopra C, Blomqvist S, Kaijalainen S, Jounio U, Juvonen R, Peitso A, et al. All known human rhinovirus species are present in sputum specimens of military recruits during respiratory infection. Viruses 2009;1:1178–89. [5] Meijer A, Benschop KS, Donker GA, van der Avoort HG. Continued seasonal circulation of enterovirus D68 in the Netherlands, 2011–2014. Euro Surveill 2014;19(42) pii: 20935. [6] Ly N, Tokarz R, Mishra N, Sameroff S, Jain K, Rachmat A, et al. Multiplex PCR analysis of clusters of unexplained viral respiratory tract infection in Cambodia. Virol J 2014;11:224. [7] Wisdom A, Leitch EC, Gaunt E, Harvala H, Simmonds P. Screening respiratory samples for detection of human rhinoviruses (HRVs) and enteroviruses: comprehensive VP4-VP2 typing reveals high incidence and genetic diversity of HRV species C. J Clin Microbiol 2009;47:3958–67. [8] Enterovirus D68 in the United States, 2014. Centers for Disease Control and Prevention [2015-01-20]. Available from: http://www.cdc.gov/non-polio-enterovirus/outbreaks/ ev-d68-outbreaks.html [9] Midgley CM, Jackson MA, Selvarangan R, Turabelidze G, Obringer E, Johnson D, et al. Severe respiratory illness associated with enterovirus D68 - Missouri and Illinois, 2014. MMWR Morb Mortal Wkly Rep 2014;63:798–9. [10] Notes from the field: acute flaccid myelitis among persons aged ⬍/ ⫽ 21 years - United States, August 1-November 13, 2014. MMWR Morb Mortal Wkly Rep 2015;63:1243–4. [11] Messacar K, Schreiner TL, Maloney JA, Wallace A, Ludke J, Oberste MS, et al. A cluster of acute flaccid paralysis and cranial nerve dysfunction temporally associated with an outbreak of enterovirus D68 in children in Colorado, USA. Lancet 2015 Jan 28 (Epub ahead of print).
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Enterovirus D68 in Sweden [12] Summary of Findings: investigation of acute flaccid myelitis in U.S. children, 2014. Centers for Disease Control and Prevention [2015-01-19]. Available from: http://www.cdc.gov/ ncird/investigation/viral/sep2014/investigation.html [13] Non-polio enterovirus - Surveillance. Public Health Agency of Canada [2015-01-20]. Available from: http://www.phacaspc.gc.ca/id-mi/vhf-fvh/enterovirus/surveillance-eng.php. [14] Helen Guiyun Li CC, Lisan Kwindt, Danuta Skowronski. Enterovirus D68. British Columbia Influenza Surveillance Bulletin 2014 (Influenza Season 2014-15, No. 3):9. [15] Enterovirus D68 (EV-D68). Alberta Health Services; 2015 [2015-02-05]. Available from: http://www.albertahealthservices.ca/10418.asp [16] Poelman R, Scholvinck EH, Borger R, Niesters HG, van Leer-Buter C. The emergence of enterovirus D68 in a Dutch University Medical Center and the necessity for routinely screening for respiratory viruses. J Clin Virol 2015;62:1–5. [17] Gimferrer L, Campins M, Codina MG, Esperalba J, Martin MD, Fuentes F, et al. First Enterovirus D68 (EV-D68) cases detected in hospitalised patients in a tertiary care university hospital in Spain, October 2014. Enferm Infec Microbiol Clin 2015 Feb 27 (Epub ahead of print). [18] Bragstad K, Jakobsen K, Rojahn AE, Skram MK, Vainio K, Holberg-Petersen M, et al. High frequency of enterovirus D68 in children hospitalised with respiratory illness in Norway, autumn 2014. Influenza Other Respir Viruses 2015;9:59–63.
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[19] Lang M, Mirand A, Savy N, Henquell C, Maridet S, Perignon R, et al. Acute flaccid paralysis following enterovirus D68 associated pneumonia, France, 2014. Euro Surveill 2014;19(44) pii: 20952. [20] Pfeiffer HC, Bragstad K, Skram MK, Dahl H, Knudsen PK, Chawla MS, et al. Two cases of acute severe flaccid myelitis associated with enterovirus D68 infection in children, Norway, autumn 2014. Euro Surveill 2015;20(10) pii: 21062. [21] Nielsen AC, Bottiger B, Midgley SE, Nielsen LP. A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience. J Virol Methods 2013;193:359–63. [22] Tiveljung-Lindell A, Rotzen-Ostlund M, Gupta S, Ullstrand R, Grillner L, Zweygberg-Wirgart B, et al. Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses. J Med Virol 2009;81:167–75. [23] Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 1999;(41):95–8. [24] Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 2010;59:307–21. [25] Tokarz R, Firth C, Madhi SA, Howie SR, Wu W, Sall AA, et al. Worldwide emergence of multiple clades of enterovirus 68. J Gen Virol 2012;93(Pt 9):1952–8.
BRIEF REPORT
Coexistence of two clades of enterovirus D68 in pediatric Swedish patients in the summer and fall of 2014
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ROBERT DYRDAK1,2, MARIA ROTZÉN-ÖSTLUND1,2, AGNETA SAMUELSON1, MARGARETA ERIKSSON3 & JAN ALBERT1,2 From the 1Department of Clinical Microbiology, Karolinska University Hospital, 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, and 3Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden
Abstract In 2014, an outbreak of enterovirus D68 (EV-D68) was observed in North America, with cases of severe respiratory illness and a possible etiological link to cases of acute flaccid paralysis. EV-D68 has also been reported from European countries, but no data from Sweden are available. This study investigated respiratory specimens collected during July–October 2014 from 30 Swedish children aged 0–9 years who were positive for enterovirus and/or rhinovirus in routine clinical PCR. Seven samples were typed as EV-D68 by VP4/VP2 sequencing. Two genetically distinct EV-D68 variants coexisted. Six viruses belonged to clade B, the variant involved in the North American outbreak, and one virus belonged to clade A. Respiratory illness was the major symptom among EV-D68 infected patients and all fully recovered. This is the first report of EV-D68 in Sweden. Considering the current epidemiological situation, genotyping and specific EV-D68 testing should be considered in patients with severe respiratory illness who test positive for enterovirus or rhinovirus in routine diagnostics.
Keywords: Enterovirus D68, pediatric, respiratory infection, VP4/VP2 sequencing
Introduction Enterovirus D68 (EV-D68) is a small, non-enveloped, single-stranded RNA virus classified within the enterovirus D species of the Picornaviridae family. Unlike other enteroviruses, but similar to rhinoviruses, EV-D68 is acid-fragile, grows poorly at 37°C, and has primarily been associated with respiratory symptoms [1]. EV-D68 has until recent years rarely been reported. In the National Enterovirus Surveillance System in the USA there were only 26 reports of EV-D68 between 1970 and 2005 [2]. In all cases but one, the virus was detected in respiratory specimens [2]. However, a study on serum samples from pregnant women in Finland collected in 1983, 1993, and 2002 showed neutralizing antibodies against the Fermon/prototype EV-D68 strain in 100% of the subjects, indicating that exposure to EV-D68 had been common [3]. In a study of Finnish military conscripts with acute respiratory infection in 2004–2005,
EV-68 was found in 3 of 32 sputum specimens [4]. Retrospective analyses have shown circulation of EV-D68 in the Netherlands during 2011–2014, and Cambodia in 2009–2012, where it was the most common virus in adults with unexplained respiratory illness [5,6]. In contrast, no EV-D68 was detected in a study of 456 respiratory samples collected in Edinburgh, Scotland in 2006 and 2007 [7]. A change in the epidemiological pattern of EV-D68 infections has occurred in 2014, with the United States experiencing a nationwide outbreak with more than 1000 cases of severe respiratory illness primarily affecting children [8,9]. In addition, EV-D68 has been detected in cases of acute flaccid paralysis, but it is still uncertain if the virus caused this condition [10–12]. Increased rates of respiratory EV-D68 infections and cases with neurological symptoms have also been reported from Canada [13–15]. In Europe, information is more limited, but an increase of EV-D68 in
Correspondence: Robert Dyrdak, Department of Clinical Microbiology, L2:02, Karolinska University Hospital, SE-171 76, Sweden. Tel: ⫹ 46 8517 72593. Fax: ⫹ 46 830 8099. E-mail: [email protected] (Received 3 March 2015 ; accepted 29 April 2015) ISSN 2374-4235 print/ISSN 2374-4243 online © 2015 Informa Healthcare DOI: 10.3109/23744235.2015.1047402
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2014 has been reported from the Netherlands [5,16], Spain [17], and Norway [18], and acute flaccid paralysis associated with EV-D68 infection was reported for one patient in France and two patients in Norway [19,20]. The epidemiology of EV-D68 in Sweden was unknown and therefore the aim of this study was to investigate the possible presence of the virus in Stockholm, Sweden.
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Materials and Methods Respiratory samples positive for enterovirus and/or rhinovirus in routine clinical PCR testing at the Department of Microbiology, Karolinska University Hospital between July and October 2014 were identified. During this period, 428 respiratory samples from pediatric patients aged 0–9 years had been submitted for analysis of enterovirus and rhinovirus. These assays were done with in-house real-time PCRs using previously described methods and primers [21,22]. Enterovirus was detected in 14 samples, rhinovirus in 80 samples, and dual reactivity in 91 samples, which was not surprising because the assays are known to sometimes cross-react. From these samples we selected 30 specimens (10 positive for enterovirus, 10 for rhinovirus, and 10 positive for both viruses). Of the 30 samples, 25 were nasopharyngeal aspirates, 2 were nasopharyngeal secretions, 1 bronchoalveolar lavage, 1 tracheal secretion, and 1 sampling from the nose. The study was approved by Regional Ethical Review board in Stockholm, Sweden (registration no. 2014/2233). For enterovirus/rhinovirus genotyping RNA was extracted using the MagAttract Virus Mini M48 Kit on a BioRobot M48 (Qiagen, Sollentuna, Sweden) for samples with threshold cycle (Ct) values below 30 in the routine real-time PCR (n ⫽ 20). For samples with higher Ct values (n ⫽ 10), RNA was manually extracted using RNeasy Lipid Tissue Mini Kit (Qiagen), which in our hands is more efficient than automated RNA extraction. RNA was eluted in 100 μl (BioRobot M48) or 40 μl (manual extraction) nuclease-free water and 7 μl of RNA template was used for nested reverse transcriptase (RT)-PCR using published primers targeting the VP4/VP2 region [7]. A one-step Superscript-Platinum Taq (Life Technologies, Stockholm, Sweden) was used for the first, outer PCR and Platinum Taq for the second, nested PCR. The PCR cycling profile was: reverse transcription for 30 min at 43°C, followed by denaturation for 2 min at 94°C, and 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 50°C, and extension for 60 s at 68°C, followed by final extension of 5 min at 68°C. The RT step was omitted in the second PCR. PCR-positive samples were purified using the Agencourt AMPure XP PCR
Purification Kit (Beckman Coulter, Bromma, Sweden), eluted with TE buffer (Invitrogen, Life Technologies, Stockholm, Sweden), and were sent for bidirectional Sanger sequencing using the inner PCR primers as sequencing primers. Sequencing was done on an ABI 3730 instrument at the KIGene sequencing facility, Karolinska Institute. The VP4/ VP2 sequences were manually aligned to reference sequences from www.picornaviridae.com using BioEdit [23]. The enterovirus species and type were determined by maximum likelihood phylogenetic trees constructed using PhyML [24] with the GTR⫹ I⫹ G substitution model. Additional EV-D68 sequences were added for detailed phylogenetic analyses. The Swedish VP4/VP2 EV-D68 sequences have been deposited in Genbank under accession numbers KP745744–KP745750. Results We investigated nasopharyngeal samples from children aged less than 10 years that had tested positive for enterovirus (n ⫽ 10), rhinovirus (n ⫽ 10) or enterovirus and rhinovirus (n ⫽ 10) in routine diagnostics at the Karolinska University Hospital between July and October 2014. Sequencing of the VP4/VP2 region was successful for 22 of 30 samples, of which RNA had been extracted manually in 4 samples and by extractor in 20 samples. Most sequencing failures occurred in samples with high Ct values in the routine real-time PCR assays, i.e. in samples with low virus content. Phylogenetic tree analysis showed that 7 of the 22 viruses were genotyped as EV-D68. These samples were collected in August and September of 2014 and were positive in the routine enterovirus PCR. Three samples also cross-reacted in the routine rhinovirus PCR. Of the remaining 15 viruses, 1 was typed as echovirus 11 within the enterovirus B species and 14 viruses were typed as different human rhinoviruses (HRV-A1, HRV-A12, HRV-A15, HRVA38, HRV-A58, HRV-A63, HRV-A80, HRV-A101, HRV-B70, HRV-B86, HRV-C6, HRV-C11, HRVC18, HRV-C45). The characteristics of the seven children with EV-D68 infection are shown in Table I. All children except one had respiratory symptoms and five had underlying diseases. Six of seven children were inpatients and two children had a short stay in the intensive care unit (ICU), but all children were discharged without remaining symptoms (apart from underlying diseases in five children). In six children no other respiratory pathogen was detected. In one child bocavirus was detected, but the virus levels were low, suggesting a carrier state rather than a symptomatic infection. Children infected with EV-D68 were
Enterovirus D68 in Sweden
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Table I. Characteristics of the seven Swedish children infected with EV-D68. Patient no. 1 2 3 4 5 6 7
Age
Gender
Symptoms
Underlying disease
9.0 years 2 months 3.8 years 3.8 years 1.2 years 3.2 years 5.3 years
F M M M M F M
Respiratory symptoms Respiratory symptoms Viral syndrome Bronchitis Viral pneumonia Respiratory symptoms Respiratory symptoms
Neuromuscular disease Recurrent wheezing No No Recurrent wheezing Recurrent wheezing Asthma
Inpatient ICU days Yes Yes No Yes Yes Yes Yes
1 0 0 0 1 0 0
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F, female; ICU, intensive care unit; M, male.
significantly older (median age 3.8 years, range 2 months to 9 years) than patients infected with rhinoviruses and echovirus 11 (median age 4 months, range 1 week to 6.8 years) (p ⫽ 0.0004, Mann-Whitney U test). Among the 23 children with other enterovirus/rhinovirus infections, respiratory disease was diagnosed in 12 patients, 7 patients had underlying conditions, and 22 were hospitalized, of which 4 were treated in the ICU. Compared with patients infected with EV-D68, patients infected with other rhinoviruses or enteroviruses less frequently had
respiratory illness as the dominating symptom, but the difference was not statistically significant (p ⫽ 0.21, Fisher’s exact test). The relationship between the seven Swedish EV-D68 viruses and international EV-D68 viruses was investigated by a more detailed phylogenetic tree analysis (Figure 1). EV-D68 clade assignment was done according to Tokarz et al. [25]. The analysis showed that six of the seven viruses belonged to clade B of EV-D68 and were closely related to each other as well as to viruses collected during the 2014
Figure 1. Phylogenetic relationships of the Swedish and international EV-D68 strains constructed using an unambiguous alignment (435 base pairs) of sequences from the VP4/VP2 region of the genome and the PhyML software with the GTR⫹ I⫹ G substitution model. Sequences from Sweden have red identifiers. The tree was midpoint rooted and the scale bar indicates the number of nucleotide substitutions per site (according to the GTR⫹ I⫹ G substitution model).
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outbreaks of EV-D68 infections in the USA and other countries. The remaining virus was genetically distinct from the six other viruses and clustered within clade A of EV-D68.
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Discussion This study for the first time reports on the presence of EV-D68 in Sweden. Although the study was small, it demonstrates that infections with EV-D68 occurred among Swedish children in the late summer and early fall of 2014. Six of the seven viruses belonged to clade B of EV-D68 and were closely related to each other and to viruses from the current North American outbreak. Interestingly, one EV-D68 virus infection with a clade A virus was also documented. Thus, two genetically distinct variants of EV-D68 coexisted in Stockholm, Sweden during the study period. The coexistence of two clades of EV-D68 means that the full epidemiological picture, such as an epidemiological link to the EV-D68 outbreak in North America, cannot be obtained by simple EV-D68 diagnostics, but requires more precise genotyping. Six of the seven EV-D68-infected children in the present study were admitted to hospital and two spent 1 day in the ICU. However, all children survived and were relatively rapidly discharged from hospital without remaining symptoms. To our knowledge there have been no reports in Sweden of increased frequency of severe respiratory infections or of unexplained acute flaccid paralysis. However, genotyping is not routinely done for respiratory samples positive for enterovirus or rhinovirus in Sweden. In addition, there has been no systematic testing for respiratory infections in children with neurological symptoms. This means that infections with EV-D68 may have gone unnoticed. Children with EV-D68 infection were significantly older than children infected with rhinoviruses and other enteroviruses. The reason for this is unclear, but it is possible that pre-existing immunity was less common to EV-D68 than to the other viruses, which would allow for infection in higher age groups. Another possibility is that EV-D68 caused more severe symptoms in older children than the other viruses and that this led to more frequent hospitalization and viral diagnostics for EV-D68-infected children in higher age groups. The study was not designed or powered to allow estimation of the prevalence and incidence of EV-D68 in Sweden, but we are currently performing a larger survey to gain better insight in this question. In light of the current outbreak of EV-D68 in North America and increasing evidence of detection also in Europe, our documentation of EV-D68 in Sweden indicates that enterovirus typing should be
considered in cases of severe respiratory illness or acute flaccid paralysis to gain more insight into the epidemiology and etiology of such infections.
Acknowledgments We would like to thank Eva Eriksson from the Department of Clinical Microbiology, for expert technical assistance, and Dr Mia Brytting from the Public Health Agency of Sweden, for valuable discussions. Declaration of interest: No funding was received. No conflicting interests.
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