the university medical center groningen is a tertiary referral center in the northern part of the netherlands .
we perform routine diagnostic real - time pcr for all patients evaluated for respiratory disease by laboratory developed tests for 17 targets , including adenovirus , bocavirus , coronavirus , enterovirus , metapneumovirus , influenza , parainfluenzavirus , rhinovirus , and respiratory syncytial virus ( 5 ) . from all enterovirus detections with a cycle threshold ( ct ) value < 31 , we sequence part of the viral protein 1 gene on a weekly basis ( 6 ) . to obtain rapid typing results in the current outbreak ,
a specific ev - d68 real - time pcr was also used ( 3 ) , when enterovirus pcr was found positive .
we identified 3 ev - d68 infections in june and 22 additional cases in july .
we found a stable seasonal variance for enterovirus and a sudden rise of enterovirus infections in july , mainly caused by ev - d68 ( figure 1 ) .
respiratory enterovirus infections , university medical center graningen , the netherlands , 2014 , 2015 , and 2016 .
the total numbers of respiratory specimens tested for enterovirus in these years during april  july were 784 , 828 , and 871 , respectively .
seventeen ( 2.2% ) of 784 were positive for enterovirus in 2014 , 5 ( 0.6% ) of /828 in 2015 , and 33 ( 3.8% ) of /871 in 2016 .
six ( 35% ) of 17 were typed as enterovirus d68 in 2014 , versus 0 of 5 in 2015 , and 25 ( 76% ) of 33 in 2016 .
the ev - d68positive specimens were nasopharyngeal swabs ( n = 8) , sputum ( n = 2 ) , and nasopharyngeal brushes ( n = 15 ) . two viral respiratory co -
infections were detected : rhinovirus a15 in patient 12 ( enterovirus ct 17 , rhinovirus ct 21 ) and parainfluenzavirus type 4 in patient 23 ( enterovirus ct 14 , parainfluenza virus 4 ct 31 ) . for 20 patients
, we obtained sequence results identifying ev - d68 ; the remaining 5 could not be typed by sequencing but were positive in our specific ev - d68 pcr .
of the 25 patients , 8 were adults and 17 were children ( table ) . in the adults , 6 of whom were transplant recipients , symptoms were mild and influenza - like . in children , however , we observed life - threatening respiratory distress , for which icu admission was necessary for 13 ( table ) .
twenty - one patients had an underlying or concurrent condition , in the children mostly pulmonary .
patient 19 acquired ev - d68 while admitted to the special care ward since birth .
this nearly 4-year - old boy had a history of headaches for 1 week and an influenza - like illness for 3 days .
he had rapidly progressing asymmetric weakness of arms and legs , bulbar paralysis , asymmetric facial paralysis , and respiratory distress for which he needed ventilatory support .
axial fluid - attenuated inversion recovery magnetic resonance imaging ( mri ) showed hyperintense nonenhancing gray matter lesions in brainstem and spinal cord .
electromyography findings supported injury on the level of the motor axon or the anterior horn of the spinal cord .
no varicella zoster virus , herpes simplex virus , enterovirus , or parechovirus were detected in the csf . in the nasopharyngeal swab
all patients in this report were treated in our tertiary referral hospital , but they originated from different regions in the north of the netherlands .
spatial epidemiology data did not suggest an epidemiologic link or outbreak ( data not shown ) . in 2 children
, ev - d68 symptoms developed > 48 hours after admission ; the source of possible nosocomial infection was not identified .
we obtained 20 sequences of part of the ev - d68 viral protein gene and aligned them with the sequences from the 2014 epidemic .
clades were assigned as described previously ( 7 ) by the neighbor - joining method by using bionumerics software 6.6 ( applied maths / biomrieux , sint - martens - latem , belgium ) .
sequence analysis ( figure 2 ) showed that the 2016 strains were closely related to sequences of the recently described subclade b3 ( 8) , represented in figure 2 by 4 sequences from china .
the nucleotide divergence was 2.1% within b3 , 5.5% between b1 and b3 , and 7.3% between b2 and b3 .
we submitted our 20 sequences from 2016 to genbank ( accession nos . kx685066kx685084 and kx710328 ) .
maximum - parsimony tree of partial viral protein 1 sequences of enterovirus d68 ( ev - d68 ) .
included are the strains obtained in the laboratory of the university medical center groningen ( groningen , the netherlands ) in 2014 ( light gray , n = 23 ) and 2016 ( black , n = 20 ) and worldwide isolates from 2014 ( dark gray , n = 73 ) .
recent strains cluster in the recently described clade b3 , with a nucleotide divergence of 2.1% within clade b3 , 5.5% to clade b1 , and 7.3% to clade b2 .
chn ,  china ; dnk ,  denmark ; esp ,  spain ; fin ,  finland ; fra ,  france ; ger ,  germany ; irl ,  ireland ; ita ,  italy ; nld ,  the netherlands ; svn ,  slovenia ; usa ,  united states .
after the upsurge of ev - d68 in our region during 2009 and 2010 , we set up routine genotypic characterization for several viral pathogens , including enterovirus and rhinovirus , which is also offered to regional referring hospitals and healthcare institutions .
sequencing results are available within 1 week , which provides clinically relevant and epidemiologic information .
this so - called regiotype strategy contributed to timely detection of ev - d68 in our hospital in 2014 , as well as in 2016 .
we recognized a sudden reappearance of ev - d68 with a sharp increase of cases that could not be explained by a change in surveillance strategy . in line with previous outbreaks ,
mostly children were affected , and those with underlying pulmonary conditions seemed at higher risk for icu admission ( 9 ) .
the number of children who needed icu admission was higher in 2016 than in our 2010 and 2014 reports ( 3,4 ) .
although dynamics of viral disease and shedding are not known for ev - d68 , we assume a role for ev - d68 in the symptoms of patients 12 and 23 , as , in line with rhinovirus infections , higher viral load might indicate symptomatic disease ( 10 ) .
evidence that ev - d68 might cause afm is increasing after recent epidemiologic investigations ( 1,11,12 ) . in patient 16 , atypical guillain - barr syndrome initially was diagnosed ; however , this diagnosis was later discarded because the electromyography results indicated motoric axon or anterior horn cell disease , and the clinical picture and mri results were in favor of afm ( 13 ) .
mri findings were subtle , and radiologic diagnosis was made only after further review and discussion of the case with the neurologists .
the absence of ev - d68 in csf is consistent with previous reports ( 1,12 ) .
sequencing results showed that the strains in our study cluster in the recently described clade b3 ( 8) . during the 2014 outbreak , most ev - d68 sequences belonged to clades b1 and b2 , although a1 and a2 were also represented ( 2,14 ) .
larger epidemiologic and genotyping studies are needed to evaluate whether the distinction within clade b is tenable and whether our clinical findings are typical for subclade b3 .
this upsurge could indicate an active ev - d68 season , as highlighted by the epidemiologic curve , with a potential increase in afm cases .
clinicians should be alert for ev - d68 , its clinical implications , and the need for appropriate diagnostics , particularly in children who are admitted with respiratory failure to the icu or with possible symptoms of afm .
 in june and july 2016 , we identified 8 adults and 17 children with respiratory enterovirus d68 infections . 
 thirteen children required intensive care unit admission because of respiratory insufficiency , and 1 had concomitant acute flaccid myelitis . 
 phylogenetic analysis showed that all of 20 sequences obtained belong to the recently described clade b3 . 
The Study
Conclusions