nasal swab specimens from outpatients with influenza - like illness ( ili ) , ari , or both were collected by sentinel physicians participating in sentinel surveillance in germany during weeks 3244 in 2014 and sent to the robert koch institute ( berlin , germany ) .
all specimens were tested in parallel for respiratory viruses , including influenza viruses a and b , rhinovirus / enterovirus , respiratory syncytial virus , adenovirus , and metapneumovirus by specific real - time reverse transcription pcrs ( technical appendix ) .
the rhinovirus / enterovirus real - time pcr detected rhinovirus at a limit of detection of 26 copies / reaction .
ev - d68 was identified at a slightly lower sensitivity of 118 copies / reaction .
rhinovirus / enterovirus  positive specimens were screened for ev - d68 by sequencing the viral protein ( vp ) 4/vp2 gene regions .
rhinovirus / enterovirus  negative specimens and samples without sequencing results were additionally analyzed by using a specific ev - d68 pcr ( 12 ) .
vp4/vp2 and vp1 regions were sequenced ( genbank accession nos . kp189380kp189403 and kp657730kp657747 ) for ev - d68positive specimens .
rhinovirus / enterovirus was detected in 44% ( 143/325 ) of the specimens ; 98 were identified as rhinovirus types a  c and 25 as ev - d68 .
the remaining 20 specimens could not be subtyped , but were negative for ev - d68 by using the specific pcr .
ev - d68 was initially detected from week 36 ( august ) through 38 ( september ) and continuously from week 41 through week 44 in october ( figure 1 ) .
detection of enterovirus d68 ( ev - d68 ) , germany , week 3244 , 2014 .
in addition to the other viruses tested , ev - d68positive specimens were screened for parainfluenza virus 14 , coronaviruses ( nl63 , oc43 , hku1 , 229e ) , and bocavirus .
none of these viruses was detected in ev - d68positive patients , which suggested that ev - d68 played a major role in causing respiratory disease .
major symptoms associated with ev - d68 infection included sudden onset , fever / shivers , cough , and sore throat ( table 1 ) .
pneumonia was diagnosed in a 7-year - old boy and a 10-year - old girl , and a 2-year - old girl was hospitalized because of obstructive bronchitis . for 11 ( 44% ) of 25 case - patients ,
a concurrent chronic condition was reported : 5 with a respiratory condition , 3 with a cardiac condition , 2 with diabetes , and 1 with a neurologic disorder .
ev - d68 was detected in 10 children and 15 adults ; 56% of these patients were male . * + , positive ; ili , influenza - like illness ;  , negative ; ari , acute respiratory infection ; na , not available . 
patients infected with ev - d68 came from different federal states in germany ; no epidemic cluster or outbreak was detected in the context of these patients .
syndromic surveillance data of corresponding sentinel practices showed only a partial coincidence of ev - d68positive patients and an increase of ari activity in the practice .
sequence analysis is not regularly performed for rhinovirus / enterovirus  positive specimens within sentinel surveillance in germany .
however , comparative data can be provided for week 36 through week 20 for the 200910 and 201011 seasons ( table 2 ) . during those seasons ,
patients with ili in 5 age groups ( < 14 , 515 , 1634 , 3560 , and > 60 years ) were investigated by using the rhinovirus / enterovirus real - time reverse transcription pcr . within the seasons ,
an average of 21% ( 198/952 for 200910 ) and 13% ( 128/1002 for 201011 ) of specimens were positive for rhinovirus / enterovirus ( table 2 ) .
at least 20% of the rhinovirus / enterovirus  positive specimens were arbitrarily chosen for sequencing ( mainly rv a , b , or c ; 1 echovirus ) , but no ev - d68 was identified . *
phylogenetic analysis of ev - d68 strains detected in germany was conducted for the vp1 and the vp4/vp2 genomic regions ( figure 2 ) .
analysis placed ev - d68 isolates from germany into major groups 1 or 3 and clustered with strains from the united states and the netherlands from 2014 , which indicated circulation of similar strains in the united states and europe .
phylogenetic analysis of selected enterovirus d68 ( ev - d68 ) strains based on nucleotide sequences of a ) partial viral protein ( vp ) 4/vp2 region ( 357 nt ) corresponding to nt 7331089 in the fermon strain ( genbank accession no .
ay426531 ) ; and b ) partial vp1 region ( 339 nt ) corresponding to nt 25212859 in the fermon strain .
trees were constructed by using maximum - likelihood estimation ( tamura 3-parameter with 5 gamma distributed rates among sites ) with 1,000 replicates through mega 5.2 ( http://www.megasoftware.net/ ) .
reference sequences were selected from the united states , countries in europe , and other regions , mainly during 20052014 .
selected reference sequences are not identical in both trees because complementary vp1 and vp4/vp2 sequence data are not available for all reference viruses .
major groups 1 , 2 , and 3 are shown as described by meijer et al .
scale bar indicate nucleotide substitutions per site . some parts of the trees are collapsed . for an expanded version of figure 2 , sees the technical appendix .
in the 2014 outbreak in the united states , 36% ( 2,600 ) of specimens were positive for ev - d68 ; children were predominantly affected .
because testing was prioritized for children with severe respiratory illness , there were probably more cases of mild infections ( 8) .
this finding might be caused by insufficient sampling of patients with ari or limited detection of ev - d68 by laboratory diagnostics ( 9 ) .
sporadic cases of neurologic disease after ev - d68 infection were reported from france and the united kingdom ( 9,10 ) .
investigation for ev - d68 has been continuously performed in the netherlands since the increase in infections in 2010 ( 13 ) .
the ili / ari sentinel system in the netherlands identified 24 ev - d68 infections in 2010 , none in 2011 , 7 in 2012 , 3 in 2013 , and 5 in 2014 ( by week 40 ) ( 13,14 ) , which probably increased toward the end of that year ( 9 ) . for the 2014 season ,
a hospital in the netherlands reported an increase of ev - d68 ; 16 patients were infected ( 12 ) .
such an increase in ev - d68 infections was already seen in 2010 at the same hospital along with an increased number of cases throughout the country ( 13 ) .
this finding increased the possibility that an increase in ev - d68 infections in primary care will also extend to increased numbers of infections in patients in secondary care .
so far , we report low ev - d68 circulation in germany : 25 sporadic cases in 2014 .
clinical patterns in patients in germany were largely determined by the ili / ari case definition for collecting specimens .
severe disease was observed in 3 children who had obstructive bronchitis and pneumonia , and 1 child required hospital care .
similarly , mild respiratory disease was predominantly observed for patients in the netherlands ( 14 ) .
however , more severe cases were detected among hospitalized children who had life - threatening respiratory illness , as described in the united states ( 6,12,14 ) .
more than half of patients with severe respiratory illness in germany and the netherlands had concurrent conditions ( 12,14 ) , which seem to be a major factor for development of severe disease after ev - d68 infection ( 6 ) .
phylogenetic analysis of ev - d68 from germany showed high similarity with current strains from the united states and the netherlands ( 12,14 ) .
these new genetic clusters reflect the evolution of ev - d68 and might be associated with an increasing activity of this virus . for an improved understanding of the factors determining local and temporal differences in respiratory disease outbreaks , continuous collection of global data by representative surveillance systems
supplemental methods used for detection of enterovirus d68associated acute respiratory infections , germany , 2014 .
 we used physician sentinel surveillance to identify 25 ( 7.7% ) mild to severe infections with enterovirus d68 ( ev - d68 ) in children and adults among 325 outpatients with acute respiratory infections in germany during august  october 2014 . 
 results suggested low - level circulation of enterovirus d68 in germany . 
 viruses were characterized by sequencing viral protein ( vp ) 1 and vp4/vp2 genomic regions . 
The Study
Conclusions
None