Enterovirus D68 infection among hospitalized children with severe acute respiratory illness in El Salvador and Panama, 2012‐2013

Abstract We assessed EV‐D68 epidemiology and phylogenetics among children aged ≤9 years hospitalized with severe acute respiratory illnesses at five sites in Panama and El Salvador during 2012‐2013. Respiratory specimens positive for enterovirus or rhinovirus were tested by real‐time RT‐PCR for EV‐D68, and partial VP1 gene sequences were determined. Of 715 enrolled children, 17 from sites in both countries were EV‐D68‐positive and commonly had a history of asthma or wheezing. Phylogenetically, 15 of 16 sequences fell into Clade B1, and one into Clade A2. The Central American EV‐D68s were closely related genetically to contemporaneous strains from North America, South America, and the Caribbean.


| ME THODS
During September-October 2012 and April-October 2013, a multisite clinical trial evaluating empiric oseltamivir treatment of hospitalized children was conducted at three hospitals in Panama (two in Panama City and one in David) and two hospitals in El Salvador (one in Santa Ana and one in San Miguel; with the San Miguel site enrolling during 2013 only). 12 Children were eligible for enrollment if they were aged ≤9 years and hospitalized <7 days after symptom onset with symptoms meeting a modified version of the World Health Organization criteria for severe acute respiratory infection (cough or sore throat plus age-specific tachypnea). Study hospitals were tertiary care facilities that provided specialized pediatric care.
Enrollment periods were selected to coincide with periods of influenza virus circulation. At enrollment, study physicians conducted a baseline clinical assessment, collected mid-turbinate nasal and throat swabs (or endotracheal aspirates for intubated children), and administered a questionnaire to the parent or guardian on participant demographics, medical history and history of illness.
Respiratory specimens were stored and tested at the Gorgas Memorial Institute for Health Studies by conventional singleplex real-time RT-PCR (rRT-PCR) using CDC protocols for influenza A and B viruses, respiratory syncytial virus, parainfluenza viruses 1-3, human metapneumovirus and rhinovirus. Specimens negative for all viruses by conventional singleplex rRT-PCR were tested by a respiratory panel pan-viral family/genus PCR (including pan enterovirus PCR) at CDC. 13 Enterovirus or rhinovirus positive specimens, by either pan enterovirus PCR or rhinovirus rRT-PCR, were tested for EV-D68 using a CDC-developed EV-D68-specific rRT-PCR assay. 14 For EV-D68-positive specimens, partial viral protein 1 (VP1) region sequences were determined. 15 and analyzed with MEGA6.0, 16 using the neighbor-joining method to infer the evolutionary history. 17 We describe frequencies of demographic and clinical characteristics of children with EV-D68 infection and all enrolled children with respiratory specimens tested for enterovirus/rhinovirus. Selected characteristics of children with and without EV-D68 infection were compared using Pearson's chi-square, Fischer's exact test, or Wilcoxon rank-sum test as appropriate. All p-values were 2-sided and evaluated for statistical significance at P < .05. Data were analyzed using STATA version 14 (STATACorp, College Station, TX).
The study protocol was approved by the national ethical review committees of El Salvador and Panama, upon which the CDC ethical review committee relied.

| D ISCUSS I ON
Mechanical ventilation at admission 1 (6) 6 (1) at three rural sites without detection at the two urban Panama City F I G U R E 1 Phylogenetic tree including enterovirus D68 (EV-D68) sequences from El Salvador and Panama. Using EV-D68 partial viral protein 1 sequences, the evolutionary history was inferred using the neighbor-joining method in MEGA 6.0 16,17 ; contemporaneous EV-D68 reference strains from GenBank were included in the analyses. EV-D68s on the tree are designated by country/state or province for some/year of detectionstrain name_GenBank accession number. Solid triangles represent sequences from El Salvador (n = 9), and solid circles represent sequences from Panama (n = 7). Bootstrap values >80% are shown. The scale bar represents genetic change in base substitutions per site