Outbreak of enterovirus D 68 of the new B 3 lineage in Stockholm , Sweden , August to September 2016

R Dyrdak 1 2 , M Grabbe 1 2 , B Hammas 1 2 , J Ekwall 3 , KE Hansson 4 , J Luthander 5 6 , P Naucler 7 8 , H Reinius 9 10 , M Rotzén-Östlund 1 2 , J Albert 1 2 1. Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden 2. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 3. Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden 4. Department of Infectious Diseases, Södersjukhuset, Stockholm, Sweden 5. Pediatric Infectious Disease Unit, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden 6. Department of Women’s and Children’s Health, Karolinska Institute, Stockholm Sweden 7. Unit of Infectious Diseases, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden 8. Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden 9. Department of Anesthesiology and Intensive Care, Akademiska University Hospital, Uppsala, Sweden 10. Department of Surgical Sciences, Uppsala University, Uppsala, Sweden

We report an enterovirus D68 (EV-D68) outbreak in Stockholm Sweden in 2016.Between 22 August and 25 September EV-D68 was detected in 74/495 respiratory samples analysed at the Karolinska University Hospital.During the peak week, 30/91 (33%) samples were EV-D68 positive.Viral protein (VP)P4/VP2 sequencing revealed that cases were caused by B3 lineage strains.Forty-four (59%) EV-D68-positive patients were children aged ≤ 5 years.Ten patients had severe respiratory or neurological symptoms and one died.
We report an outbreak of enterovirus D68 (EV-D68) infections in Stockholm, Sweden in late August and September of 2016 caused by the newly described B3 lineage [1].

Patients, samples and routine diagnostics for respiratory viruses
The main study was based on respiratory samples analysed at the Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden, between 22 August and 25 September 2016 (n = 495; 183 nasopharyngeal aspirates, 232 nasopharyngeal swabs, 77 lower respiratory tract samples, 3 unspecified respiratory samples).The laboratory provides diagnostic services to six of seven major hospitals and approximately half of outpatient care in the Stockholm county (2.2 million inhabitants).Most samples (480 of 495) were from the catchment area and collected as part of routine diagnostics from inpatients and, to a lesser degree, outpatients.Fifteen samples were referred from other counties.For comparison, results from routine EV and rhinovirus diagnostics from the Karolinska University Hospital in 2014, 2015, and the remaining part of 2016 up to 13 November were also analysed.
EV, rhinovirus and 10 other respiratory viruses were diagnosed using in-house real-time polymerase chain reactions (PCR)s [1].The PCRs for EV and rhinovirus cross-react because the viruses are closely related.Based on results from extensive validation including sequencing, samples with dual reactivity for EV and rhinovirus were classified as rhinovirus if the PCR cycle threshold (Ct)-value for rhinovirus was > 3 lower than the Ct-value for EV and otherwise as EV.Influenza A, influenza B and respiratory syncytial virus were diagnosed using the commercial Simplexa system [2].The study was approved by Regional Ethical Review board in Stockholm, Sweden (registration number 2016/2004-32).

Enterovirus D68 polymerase chain reaction and sequencing
A real-time EV-D68 PCR was introduced in the late summer of 2016 and was based on the primers and probe of Piralla et al. [3] and used 5 µL of extracted RNA, 5 µL TaqPath 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific, Stockholm, Sweden), 100 nM of primers and probe in a total volume of 20 µL.An ABI7500 FAST Real Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Stockholm, Sweden) was used with the following cycling profile: 2 min at 25 °C, 15 min at 50 °C, 2 min at 95 °C, and 45 cycles of denaturation for 10 s at 95 °C, annealing for 30 s at 60 °C.a Enterovirus D68 data are not available from 25 September onwards, after which the test was only done on demand.

Figure 2
Maximum likelihood phylogenetic tree constructed using enterovirus D68 viral protein (VP)4/VP2 sequences (435 bp) from Stockholm, Sweden and relevant GenBank sequences Sequencing of the viral protein (VP)4/VP2 region of the EV/rhinovirus genome was performed with an in-house protocol and primers by Wisdom et al. [4,5].EV-D68 sequences were deposited in GenBank under accession numbers KY215827-69*.The EV/rhinovirus species and type were determined by maximum likelihood phylogenetic trees constructed using Molecular Evolutionary Genetics Analysis (MEGA) 7.0.18(GTR + I + G model), which included reference sequences available at www. picornaviridae.comand EV-D68 sequences that were downloaded from GenBank after a search using basic local alignment search tool (BLAST).

Description of the enterovirus D68 outbreak in Stockholm in the early autumn of 2016
Of the 495 respiratory samples obtained in the main study period between 22 August to 25 September, 72 were positive for rhinovirus alone while 122 (>25%) reacted as EV positive.Among these 122 samples, 21 tested positive for EV alone, and 101 were dually reactive for both rhinovirus and EV.Based on the analysis of Ct-values, 67 of the 101 dually reactive samples most likely contained EV alone, while 34 of these samples likely bore only rhinovirus.Thus a total of 88 samples were classified as EV positive and 106 samples were classified as rhinovirus positive (Figure 1A).The proportion of EV positive samples during the study period in 2016 (18%; 88/495) was significantly higher than the corresponding period in 2015 (2%; 9/366; p<0.001,Fisher exact test) and also higher than in 2014 (15%; 49/321) (Figure 1B).
A total of 149 respiratory samples from the study period were analysed with EV-D68 PCR.These included a subset of 34 samples that had tested positive for rhinovirus alone in earlier PCRs, and 115 samples with available material, among the 122 initially appearing as EV positive; 74 samples were positive for EV-D68.
In the week of 29 August (week 35), 33% (30/91) of all respiratory samples were positive for EV-D68 (Figure 1A).EV-D68-negative samples usually had an indication of rhinovirus based on the Ct-value for rhinovirus being > 3 lower than the Ct-value for EV.In 20 of these samples rhinovirus was verified by VP4/VP2 sequencing.The 34 samples that had tested only positive for rhinovirus in prior PCRs were found negative by EV-D68 PCR.
In Figure 1A the two curves depicting the variations with time of the proportions of EV-and EV-D68-positive samples among all respiratory samples analysed during the study period, have almost identical trajectories.This justifies the classification EV and rhinovirus positive samples based on Ct-values.The Figure also indicates that almost all EV infections in the main study period were caused by EV-D68.After 25 September, specific EV-D68 diagnostics were only done on demand of physicians and the proportion of EV-positive samples remained at a lower level up to 13 November, suggesting that EV-D68 activity was likely low in October and early November.

The outbreak was caused by the new B3 lineage of enterovirus D68
VP4/VP2 sequencing was attempted on 80 samples from the study period (57 EV-D68 positive and 23 EV-D68 negative).Figure 2 shows that all successfully sequenced EV-D68 PCR positive samples from 2016 (n = 43) belonged to the recently described B3 lineage of EV-D68 [6].Within the B3 lineage, 41 of 43 Swedish sequences from 2016 formed a tight cluster together with unpublished Genbank sequences from the United States (US) collected in 2016.

Discussion
In 2014 EV-D68 emerged worldwide [7].The emergence received high attention by public health authorities because of its magnitude and the clinical presentation of some patients who displayed severe respiratory and neurological symptoms, including acute flaccid paralysis [7][8][9][10].
There are indications that EV-D68 may be resurging in 2016 [11,12], but due to lack of systematic surveillance the true disease burden is unclear [7,11] The outbreak was caused by closely related strains of the recently described B3 lineage [6].Available data indicate that the B3 lineage arose recently in the evolution of EV-D68 and is actively spreading in parts of Europe [12] and the US during the 2016 season (unpublished GenBank sequences).It is unclear if the apparent epidemiological success of this lineage in 2016 is due antigenic drift or if the risk of severe disease differs from other EV-D68s, such as the B1 lineage that caused the worldwide outbreak in 2014.
In a recent rapid risk assessment the European Centre for Disease Prevention and Control (ECDC) stated that the increased numbers of EV-D68 (and EV-A71) detections reinforce the need for vigilance for EV infections, especially cases that present with more severe clinical syndromes [11].This appears insightful in light of the recent outbreak in Stockholm.

Addendum
*The GenBank accession numbers were added on 23 November 2016.

Table
Characteristics of 10 enterovirus D68 infected patients with severe symptoms, Stockholm, Sweden, 22 August-25 September 2016 a Patient diagnosed in October, outside of the main study period from 22 August to 25 September.