Severe enterovirus 68 respiratory illness in children requiring intensive care management

Journal of Clinical Virology 70 (2015) 77–82

Contents lists available at ScienceDirect

Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv

Severe enterovirus 68 respiratory illness in children requiring intensive care management Jennifer E. Schuster a,∗,1 , Jenna O. Miller b,1 , Rangaraj Selvarangan c , Gina Weddle a , Marita T. Thompson b , Ferdaus Hassan c , Shannon L. Rogers d , M. Steven Oberste d , W. Allan Nix d , Mary Anne Jackson a a

Division of Infectious Diseases, Children’s Mercy Hospital, Kansas City, MO, USA Division of Critical Care, Children’s Mercy Hospital, Kansas City, MO, USA c Division of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, USA d Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA b

a r t i c l e

i n f o

Article history: Received 20 June 2015 Accepted 16 July 2015 Keywords: Acute respiratory tract infection Asthma Enterovirus EV-D68 Intensive care unit Respiratory virus

a b s t r a c t Background: Enterovirus 68 (EV-D68) causes acute respiratory tract illness in epidemic cycles, most recently in Fall 2014, but clinical characteristics of severe disease are not well reported. Objectives: Children with EV-D68 severe respiratory disease requiring pediatric intensive care unit (PICU) management were compared with children with severe respiratory disease from other enteroviruses/rhinoviruses. Study design: A retrospective review was performed of all children admitted to Children’s Mercy Hospital PICU from August 1-September 15, 2014 with positive PCR testing for enterovirus/rhinovirus. Specimens were subsequently tested for the presence of EV-D68. We evaluated baseline characteristics, symptomatology, lab values, therapeutics, and outcomes of children with EV-D68 viral infection compared with enterovirus/rhinovirus-positive, EV-D68-negative children. Results: A total of 86 children with positive enterovirus/rhinovirus testing associated with respiratory symptoms were admitted to the PICU. Children with EV-D68 were older than their EV-D68-negative counterparts (7.1 vs. 3.5 years, P = 0.01). They were more likely to have a history of asthma or recurrent wheeze (68% vs. 42%, P = 0.03) and to present with cough (90% vs. 63%, P = 0.009). EV-D68 children were significantly more likely to receive albuterol (95% vs. 79%, P = 0.04), magnesium (75% vs. 42%, P = 0.004), and aminophylline (25% vs. 4%, P = 0.03). Other adjunctive medications used in EV-D68 children included corticosteroids, epinephrine, and heliox; 44% of EV-D68-positive children required non-invasive ventilatory support. Conclusions: EV-D68 causes severe disease in the pediatric population, particularly in children with asthma and recurrent wheeze; children may require multiple adjunctive respiratory therapies. © 2015 Elsevier B.V. All rights reserved.

1. Background

Abbreviations: EV-D68, enterovirus 68; PICU, pediatric intensive care unit; LRTI, lower respiratory tract illness; ICU, intensive care unit; CDC, Centers for Disease Control and Prevention; RPP, respiratory pathogen panel; CMH, Children’s Mercy Hospital; NIV, non-invasive ventilation; IQR, interquartile range; ANC, absolute neutrophil count; ALC, absolute leukocyte count; RAD, reactive airway disorder; AFM, acute flaccid myelitis. ∗ Corresponding author at: Children’s Mercy Hospital, 2401 Gillham Road, Kansas City, MO 64108, USA. Fax: +1 816 346 1328. E-mail address: [email protected] (J.E. Schuster). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jcv.2015.07.298 1386-6532/© 2015 Elsevier B.V. All rights reserved.

Enterovirus 68 (EV-D68) was identified from oropharyngeal swabs of 4 children hospitalized with acute lower respiratory tract illness (LRTI) in 1962 [1]. EV-D68 has features of both enteroviruses and rhinoviruses and is associated with respiratory symptoms [2,3]. Many multiplex PCR assays used in clinical practice do not distinguish between the two species, so the manifestations and severity of EV-D68 have not been well characterized. Previous epidemiologic studies have been primarily retrospective [4–7], and rates of detection have been <1% [7,8]. Although the majority of infections were in children [8,9], pediatric cohorts were small, usually less than 15 patients [5,10,11]. In mixed adult and pediatric cohorts,

78

J.E. Schuster et al. / Journal of Clinical Virology 70 (2015) 77–82

underlying medical conditions were common [12,13]. Intensive care unit (ICU) stays were rare [5,11,14], and in reports of severe disease, cohorts contained less than 5 patients [4,13]. EV-D68 infections generally occur in the late summer-early fall months and in unpredictable epidemic cycles [5,7,14–16]. Prior to 2006, EV-D68 was rarely identified. The National Enterovirus Surveillance System, a voluntary passive surveillance mechanism through the Centers for Disease Control and Prevention (CDC), reported 26 cases from 1970 to 2005. EV-D68 accounted for 0.1% of reported enteroviruses during that period [16]. Between 2008 and 2010, multiple countries reported EV-D68 outbreaks or clusters [4,7,10,13,17]. After 2010, EV-D68 was again rarely reported until Fall 2014 when a resurgence of EV-D68 disease was identified in the United States and Europe [18–20].

2. Objective We aim to describe severe EV-D68 disease, including at-risk populations, presenting symptoms, and extent of intensive therapies used compared with children with severe disease from other enteroviruses/rhinoviruses. We report the largest cohort of pediatric EV-D68 disease to date, and the first to focus on children requiring pediatric ICU (PICU) stay.

Table 1 Demographic characteristics of PICU children with EV-D68 and with other enteroviruses/rhinoviruses.

Median age (IQR) – yearsb Male sex – no. (%) Race or ethnic group – no. (%)c White Black Hispanic or Latino Other/unknown High risk condition – no. (%) Asthma Recurrent wheeze Asthma or recurrent wheeze Neurologic Prematurity Cardiac None

EV-D68 positive N = 59

EV-D68 negative N = 24

P valuea

7.1 (3.0–11.5) 39 (66)

3.5 (0.9–7.8) 18 (75)

0.01 0.43 0.28

25 (42) 20 (34) 8 (14) 6 (10)

14 (58) 5 (21) 1 (4) 4 (17)

30 (51) 10 (17) 40 (68) 5 (9) 4 (7) 1 (2) 12 (20)

8 (33) 2 (8) 10 (42) 5 (21) 5 (21) 2 (8) 6 (25)

0.15 0.49 0.03 0.14 0.11 0.20 0.64

a P values were calculated by Pearson chi-square or Fisher’s exact test, except for median age, which was calculated by Wilcoxon rank-sum test. b IQR denotes interquartile range. c Race and ethnic group were determined according to parental/guardian report in the medical record.

3.4. Statistical analysis 3. Study design 3.1. Study subjects An increase in emergency department visits and hospital admissions associated with severe respiratory disease was noted on August 15, 2014 at Children’s Mercy Hospital (CMH). A case definition was established on August 21, and respiratory pathogen panel (RPP) (Biofire Inc, Salt Lake City, Utah) testing was recommended for all inpatient children with increased work of breathing requiring supplemental oxygen or continuous albuterol. This assay does not distinguish between human enteroviruses and rhinoviruses [21]. Patients, aged 0–17 years, admitted to CMH with multiplex RPP testing positive for enterovirus/rhinovirus from August 1 to September 15, 2014 were retrospectively identified. Testing for EV-D68 was done after the patient encounter, so results were unavailable to the clinician. Patients requiring hospitalization in the PICU were identified from the larger cohort by chart review. CMH’s Institutional Review Board approved the study.

Nominal variables were described using total number and percentage with Pearson Chi-square test, or Fisher’s exact test, to determine significance between the groups. Continuous variables were described by median with interquartile range and Wilcoxon rank-sum test. All statistics were performed in SPSS (versions 18.0 and 20.0). 4. Results From August 1 to September 15, 2014, 562 children were admitted to CMH, had positive enterovirus/rhinovirus testing, and had specimens available for EV-D68 testing. 61/341 (17.9%) EV-D68 positive (EV-D68+) children required PICU management compared with 34/221 (15.4%) children with non-EV-D68 enteroviruses/rhinoviruses (EV-D68-). Of the 95 PICU children, 10 EV-D68- children and 2 EV-D68+ children were excluded because they required PICU management for non-respiratory reasons. Thus, 83 children were included in the final analysis: 59 EV-D68+ children and 24 EV-D68- children (Fig. 1).

3.2. Virologic testing

4.1. Baseline characteristics

Prior to the development of real-time RT-PCR for EV-D68 [22], samples were sequenced based on the enterovirus VP1 region at the CDC [23]. Once an EV-D68-specific RT-PCR method was developed, the remaining samples were tested by RT-PCR only at CMH. Total RNA was extracted [24] and tested per CDC protocol with minor modification of the probe to use 5-Cy5 dye [25]. All primers and probes were purchased from IDT DNA Inc (Coralville, IA).

EV-D68 + patients were significantly older than EV-D68patients (7.1 vs. 3.5 years, P = 0.01) (Table 1). The majority of patients were male. No statistically significant racial differences were noted. Approximately, 55% of both groups had publicly funded insurance. Among EV-D68+ patients, 40 (68%) had a history of asthma or recurrent wheeze, compared with 10 (42%) of EV-D68- children, P = 0.03. Other chronic medical conditions were not significantly different between the two groups. Twelve (20%) EV-D68+ patients had no reported high-risk condition. No differences in household smokers or daycare/school exposure were noted.

3.3. Patient data Chart review was performed, and data were entered into a standardized document in REDCap [26]. Laboratory and radiographic values were the first documented results, from either CMH or the transferring hospital. Treatments included only therapies administered at CMH. Non-invasive ventilation (NIV) was defined as high flow nasal cannula >5 l/min, continuous positive airway pressure, or biphasic positive airway pressure.

4.2. Clinical signs and symptoms Length of illness prior to presentation was not different between EV-D68+ and EV-D68- children (2 vs. 1.5 days, P = 0.83). Respiratory symptoms were prominent in both groups (Table 2). Cough was the only parent-reported symptom significantly different between EV-

J.E. Schuster et al. / Journal of Clinical Virology 70 (2015) 77–82

79

Fig. 1. Number of specimens positive for enterovirus/rhinovirius in children requiring PICU management.

Table 2 Parental report of symptoms and clinical signs of PICU children with EV-D68 and with other enteroviruses/rhinoviruses. EV-D68 positive N = 59

EV-D68 negative N = 24

P valuea

Symptom – no. (%)b Asthma exacerbation or wheezing Chest pain Cough Cyanosis Diarrhea Dyspnea/increased work of breathing Fever Nasal congestion Rhinorrhea Seizures Sore throat Vomiting

29 (49) 7 (12) 53 (90) 1 (2) 1 (2) 55 (93) 24 (41) 26 (44) 22 (37) 1 (2) 9 (15) 14 (24)

10 (42) 3 (13) 15 (63) 3 (13) 1 (4) 20 (83) 10 (42) 12 (50) 7 (29) 2 (8) 1 (4) 9 (38)

0.54 1.00 0.009 0.07 0.50 0.22 0.93 0.62 0.48 0.20 0.27 0.20

Sign – no. (%) Feverc Hypoxiad Retractionse Wheezinge

8 (14) 42 (71) 49 (83) 44 (75)

5 (21) 20 (83) 18 (66) 15 (63)

0.51 0.28 0.10 0.27

in both groups presented with hypoxia (71% vs. 83%, P = 0.28). Initial physical examination findings reflected parental report of increased work of breathing. Retractions (83% vs. 67%, P = 0.10) and wheezing (75% vs. 63%, P = 0.27) were more common in EV-D68+ children although this was not statistically significant. 4.3. Laboratory and radiographic findings Approximately, half of the children in both groups had laboratory work performed (Table 3). EV-D68+ children had a significantly higher absolute neutrophil count (ANC) than EV-D68- children (10.2 vs. 5.6 × 103 /␮l, P = 0.01). C-reactive protein was mildly elevated in both groups, although it was infrequently obtained. The majority of children in both groups had chest radiographs (92% vs. 92%, P = 1.00), and most of the radiographs had abnormalities (67% vs. 82%, P = 0.19). Abnormal findings included peribronchial/perihilar infiltrates, lobar opacification, hyperexpansion, and atelectasis. Specific findings were not significantly different between the two groups. 4.4. Evaluation for secondary infections

a

P values were calculated by Pearson chi-square or Fisher’s exact test. Percentages do not total 100% since many children had more than one complaint. Symptoms obtained from parental report. c Fever defined as a temperature ≥38.5 ◦ C during hospitalization. d Hypoxia defined as oxygen saturation <95% on initial measurement. e Presence of retractions and wheezing were taken from initial physical exam. b

D68+ and EV-D68- children (90% vs. 63%, P = 0.009). Complaints of dyspnea and/or increased work of breathing were common (93% vs. 83%, P = 0.22). No differences in upper respiratory symptoms were noted. Vomiting, often post-tussive, was not significantly different (24% vs. 38%, P = 0.20), and diarrhea was uncommon. Although parental report of fever was common (approximately 40%), only 14% of EV-D68+ and 21% of EV-D68-children had a temperature ≥38.5 ◦ C during hospitalization. The majority of children

Testing for bacterial pathogens was performed in both groups. Urine culture was obtained in 3 (5%) EV-D68+ children compared with 5 (21%) EV-D68- children, P = 0.04. 14 (24%) of EV-D68+ children vs. 8 (33%) of EV-D68- children had a blood culture obtained, P = 0.37, and cerebrospinal fluid cultures were rarely obtained (0% vs. 8%, P = 0.08). No cultures were consistent with a secondary bacterial infection. Adenovirus was detected by RPP from 1 patient in each group. No other pathogens were identified from RPP testing. 4.5. Treatment Respiratory interventions were numerous and intensive. Most therapies were directed at asthma-like symptoms, and bronchodilator use was common (Table 4). Albuterol was used in

80

J.E. Schuster et al. / Journal of Clinical Virology 70 (2015) 77–82

Table 3 Initial laboratory findings of PICU children with EV-D68 and with other enteroviruses/rhinoviruses.

Median white blood cell count (IQR) – 103 /␮l Median absolute neutrophil count (ANC) (IQR) – 103 /␮l Median absolute lymphocyte count (ALC) (IQR) – 103 /␮l Median hemoglobin (IQR) – gm/dl Median C-reactive protein (IQR) – mg/dl a b c

EV-D68 positive N = 26a

EV-D68 negative N = 14b

P valuec

14.5 (10.4–18.8) 10.2 (7.0–14.2) 1.7 (1.1–2.7) 12.0 (11.0–13.6) 2.2 (1.4–4.3)

12.3 (8.5–13.6) 5.6 (3.5–10.0) 2.0 (1.2–4.1) 11.6 (10.3–12.4) 1.6 (0.8–3.0)

0.89 0.01 0.36 0.25 0.32

N = 23 for ANC and ALC. N = 9 for C-reactive protein. N = 13 for ANC, ALC, and hemoglobin. N = 6 for C-reactive protein. P values were calculated by Wilcoxon rank-sum test.

Table 4 Therapies used in PICU children with EV-D68 and with other enteroviruses/rhinoviruses. All children

Oxygen supplementation – no. (%) Hours of oxygen (IQR) Albuterol Continuous albuterol Hours of continuous albuterol – (IQR) Corticosteroids – no. (%) Magnesium – no. (%) Aminophylline – no. (%) Epinephrineb – no. (%) Heliox – no. (%) Ventilatory assistance Non-invasive ventilation – no. (%) Conventional ventilation – no. (%) a b

Children with asthma or recurrent wheeze a

P valuea

EV-D68 positive N = 59

EV-D68 negative N = 24

P value

EV-D68 positive N = 40

EV-D68 negative N = 10

58 (98) 54 (17–96) 56 (95) 50 (89) 8 (5–16) 51 (86) 44 (75) 15 (25) 14 (24)

24 (100) 75 (19–117) 19 (79) 13 (68) 5 (4–8) 16 (67) 10 (42) 1 (4) 2 (8)

1.00 0.40 0.04 0.06 0.05 0.06 0.004 0.03 0.13

40 (100) 52 (17–92) 40 (100) 38 (95) 10 (5–21) 40 (100) 36 (90) 15 (38) 13 (33)

10 (100) 49 (13–101) 10 (100) 9 (90) 7 (4–8) 10 (100) 6 (60) 0 (0) 1 (10)

6 (10)

0 (0)

0.18

6 (15)

0 (0)

0.33

26 (44)

9 (38)

0.58

17 (43)

4 (40)

1.00

4 (7)

4 (17)

0.22

1 (2.5)

0 (0)

1.00

0.98 0.50 0.02 0.04 0.02 0.25

Variables were analyzed by Pearson Chi-square or Fisher’s exact test. Hours on oxygen and albuterol were analyzed by Wilcoxon rank-sum test. Subcutaneous or intramuscular.

more EV-D68+ patients (95% vs. 79%, P = 0.04) as was continuous albuterol (89% vs. 68%, P = 0.06). EV-D68+ children received continuous albuterol for a median of 8 h (range 1–69) compared with 5 h (range 1-10) in EV-D68- children, P = 0.05. More EV-D68+ children received magnesium (75% vs. 42%, P = 0.004), and aminophylline (25% vs. 4%, P = 0.03). Corticosteroids (86% vs. 67%, P = 0.06), subcutaneous or intramuscular epinephrine (23% vs. 7%, P = 0.18), and heliox (10% vs. 0%, P = 0.13) were also administered more often in EV-D68+ children although this was not statistically significant. EV-D68+ children with a history of asthma or recurrent wheeze received more hours of continuous albuterol, more magnesium, and more aminophylline, than their EV-D68- children with a history of asthma or recurrent wheeze. Several children received at least one dose of antibiotics (25% vs. 42%, P = 0.14). Third-generation cephalosporins were the most frequently prescribed, followed by aminopenicillins and vancomycin in both groups. The majority of children in both groups required oxygen (98% vs. 100%, P = 1.00) for a median of 54 vs. 75 h. NIV was utilized in 44% of EV-D68+ children compared with 38% of EV-D68- children, P = 0.58. A smaller proportion of children required conventional ventilation (7% vs. 17%, P = 0.22). No child in either group required high frequency oscillating ventilation or extracorporeal mechanical oxygenation. Specific ventilatory support was not significantly different between the two groups.

4.6. Outcome Neither median length of PICU stay (23 vs. 26 h, P = 0.61) nor median length of hospital stay (82 vs. 114 h, P = 0.10) was significantly different between the groups. No deaths occurred in either

group during hospitalization. EV-D68+ children were significantly less likely to be re-admitted within 30 days (1.7% vs. 16.7%, P = 0.02). No EV-D68+ patient was re-admitted for acute flaccid myelitis (AFM).

5. Discussion In August 2014, we identified an outbreak of severe respiratory infection in children, which was confirmed to be caused by EV-D68 [18]. The need for ICU management of children was uncommonly reported in previous EV-D68 outbreaks, and descriptive literature provided little data to highlight specific clinical features, potential high-risk populations, or effective therapies. To our knowledge, this study is the largest pediatric cohort of EV-D68 disease and is the first to characterize severe infection in comparison to other enterovirus/rhinoviruses. EV-D68+ children were older than those infected with other enteroviruses or rhinoviruses, and they were more likely to have a history of asthma or recurrent wheezing. Other chronic medical conditions were uncommon, and unlike previous reports, none of our patients with severe disease were immunocompromised [27]. Importantly, 20% of our critically ill patients with EV-D68 had no chronic medical conditions, highlighting the significant disease severity of EV-D68 in previously healthy children. Signs of respiratory distress were common, whereas fever was uncommon. Laboratory values were generally not different between the groups, and abnormal chest radiographs were common, highlighting the potential role for an EV-D68-specific rapid diagnostic test [8,12]. Children with severe EV-D68 infections received asthmatargeted therapeutics, of which bronchodilators and corticosteroids

J.E. Schuster et al. / Journal of Clinical Virology 70 (2015) 77–82

are the foundation [28]. However, studies have shown that asthma therapy varies among institutions [29,30], including in the PICU [31]. In our EV-D68+ cohort, albuterol, corticosteroids, magnesium, and aminophylline were commonly used, even in children without a history of asthma. Magnesium and aminophylline were more commonly used in our asthmatic EV-D68+ patients compared with asthmatic EV-D68- patients, suggesting a more severe and refractory disease. Additionally, rates of use of these drugs were higher compared to national data of PICU asthma exacerbations [32]. Many of the therapies used, including aminophylline and heliox, cannot be administered outside of an intensive care setting. Therefore, practitioners should be aware EV-D68+ children may need to be managed in a facility with intensive care services. Although deaths in association with the current EV-D68 outbreak were reported [33], we had no mortality at our institution. Practitioners should be highly suspicious for EV-D68 infection in school-aged children with acute onset wheezing and hypoxia that is resistant to typical asthma interventions, especially when presenting outside of the winter respiratory virus season and should consider aggressive bronchospasm therapy. The role of steroids causing fulminant disease and fatal outcomes in children with enterovirus 71 neuroinvasive disease has been investigated. Additionally, past reports of Hopkins’ syndrome, first described in the early 1970s, raised concern for the development of acute flaccid paralysis in conjunction with or shortly after a steroid-treated asthma exacerbation [34–36]. However, in the last 30 years, inhaled and oral corticosteroids have been established as the mainstay of therapy for children with asthma. It is interesting to note that concurrent with the EV-D68 outbreak, there were reports of AFM cases in the United States, many of whom had antecedent respiratory tract symptoms and/or positive testing for EV-D68 [37]. To date, an etiology has not been delineated, and no association with steroid use during the acute phase of respiratory illness has been observed. In our cohort of EV-D68+ children with severe disease, the majority of patients received corticosteroids, as an intervention that is supported by evidence-based asthma treatment guidelines. We had no cases of AFM in any of the EV-D68+ children who required hospitalization. Despite the severity of disease in our patient population, circulating Kansas City outbreak strains cluster with previously sequenced EV-D68 strains, with only small genome changes [38]. Other respiratory viruses demonstrate year-to-year variation in incidence and hospitalization rates [39,40]. Therefore, we hypothesize that EV-D68 may have circulated previously in the United States and caused mild respiratory tract symptoms or was present at a lower incidence. Seroprevalence studies could shed some light on this possibility. Without routine use of a specific EV-D68 test, mild disease may have previously gone unnoticed, and sporadic cases of severe asthma may not have been recognized to have an infectious trigger. PICU care is infrequently needed for children admitted with asthma [41,42], and in our cohort, viral or bacterial co-infection was rare, indicating that disease severity was likely related to EV-D68. Without licensed antivirals, supportive care with asthma-directed therapies is crucial. We may have overlooked cases that occurred prior to August 1, and cases in which testing was not ordered. We stopped data collection on September 15, after the peak of the outbreak, in order to analyze our cohort, and we did not include children who tested positive after this date. We reviewed charts to exclude children who had another plausible cause for their respiratory symptoms, but some children may have had unrecognized conditions contributing to disease severity. Information was collected retrospectively, and we relied on data from the medical chart. Therefore, history and symptoms were dependent on passive parental report and provider documentation. Lastly, children who initially presented to outside facilities likely received therapeu-

81

tics, which could have changed presenting physical examination findings. Future research should be directed at improving the knowledge of EV-D68 epidemiology, disseminating specific EV-D68 laboratory tests [12,22], and identifying EV-D68-specific therapies. Prospective follow-up is needed to determine the long-term outcomes of children with EV-D68 infection. Due to the severity of infection and bronchospasm symptoms, we hypothesize that in young children, EV-D68 may subsequently be associated with the development of asthma, similar to other respiratory viruses [43]. We report the largest cohort of pediatric EV-D68 disease and the first to focus on children with severe EV-D68 disease requiring PICU stay compared with severe infection from other enteroviruses/rhinoviruses. Our study highlights the association of EV-D68 with severe respiratory disease in older children with a history of asthma and wheeze. Despite variations in the treatment of asthma and the purported association of both enteroviruses and corticosteroids with Hopkins’ syndrome, therapies targeting bronchospasm appear to be of utmost importance to children with severe EV-D68. Additionally, we report a significant cohort of children with no prior wheezing history but may require ICU care and benefit from asthma-directed therapies. In our experience, adjunctive asthma therapies appear to be safe and highly effective for such patients. Practitioners should proactively provide asthma action plans for asthmatic children and be cognizant of the potential for severe EV-D68 disease in such patients. Competing interests None declared. Ethical approval Children’s Mercy Hospital Institutional Review Board approved this study. Funding This work was funded in part by federal appropriations from the Emerging Infections line item to the Department of Health and Human Services. References [1] J.H. Schieble, V.L. Fox, E.H. Lennette, A probable new human picornavirus associated with respiratory diseases, Am. J. Epidemiol. 85 (1967) 297–310. [2] S. Blomqvist, C. Savolainen, L. Raman, M. Roivainen, T. Hovi, Human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features, J. Clin. Microbiol. 40 (2002) 4218–4223. [3] M.S. Oberste, K. Maher, D. Schnurr, M.R. Flemister, J.C. Lovchik, H. Peters, et al., Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses, J. Gen. Virol. 85 (2004) 2577–2584. [4] T. Imamura, N. Fuji, A. Suzuki, R. Tamaki, M. Saito, R. Aniceto, et al., Enterovirus 68 among children with severe acute respiratory infection, the Philippines, Emerg. Infect. Dis. 17 (2011) 1430–1435. [5] L.M. Jacobson, J.T. Redd, E. Schneider, X. Lu, S.W. Chern, M.S. Oberste, et al., Outbreak of lower respiratory tract illness associated with human enterovirus 68 among American Indian children, Pediatr. Infect. Dis. J. 31 (2012) 309–312. [6] I.L. Lauinger, J.M. Bible, E.P. Halligan, E.J. Aarons, E. MacMahon, C.Y. Tong, Lineages, sub-lineages and variants of enterovirus 68 in recent outbreaks, PLoS One 7 (2012) e36005. [7] A. Meijer, S. van der Sanden, B.E. Snijders, G. Jaramillo-Gutierrez, L. Bont, C.K. van der Ent, et al., Emergence and epidemic occurrence of enterovirus 68 respiratory infections in The Netherlands in 2010, Virology 423 (2012) 49–57. [8] A. Piralla, A. Girello, M. Grignani, M. Gozalo-Marguello, A. Marchi, G. Marseglia, et al., Phylogenetic characterization of enterovirus 68 strains in patients with respiratory syndromes in Italy, J. Med. Virol. 86 (2014) 1590–1593. [9] A. Meijer, K.S. Benschop, G.A. Donker, H.G. van der Avoort, Continued seasonal circulation of enterovirus D68 in the Netherlands, 2011–2014, Euro Surveillance 19 (42) (2014), pii:20935.

82

J.E. Schuster et al. / Journal of Clinical Virology 70 (2015) 77–82

[10] A. Kaida, H. Kubo, J. Sekiguchi, U. Kohdera, M. Togawa, M. Shiomi, et al., Enterovirus 68 in children with acute respiratory tract infections, Osaka, Japan, Emerg. Infect. Dis. 17 (2011) 1494–1497. [11] F. Renois, A. Bouin, L. Andreoletti, Enterovirus 68 in pediatric patients hospitalized for acute airway diseases, J. Clin. Microbiol. 51 (2013) 640–643. [12] R. Poelman, E.H. Scholvinck, R. Borger, H.G. Niesters, C. van Leer-Buter, The emergence of enterovirus D68 in a Dutch University Medical Center and the necessity for routinely screening for respiratory viruses, J. Clin. Virol. 62 (2015) 1–5. [13] J. Rahamat-Langendoen, A. Riezebos-Brilman, R. Borger, R. van der Heide, A. Brandenburg, E. Scholvinck, et al., Upsurge of human enterovirus infections in patients with severe respiratory tract infections, J. Clin. Virol. 52 (2011) 103–106. [14] P. Linsuwanon, J. Puenpa, K. Suwannakarn, V. Auksornkitti, P. Vichiwattana, S. Korkong, et al., Molecular epidemiology and evolution of human enterovirus serotype 68 in Thailand, 2006–2011, PLoS One 7 (2012) e35190. [15] T. Imamura, A. Suzuki, S. Lupisan, M. Okamoto, R. Aniceto, R.J. Egos, et al., Molecular evolution of enterovirus 6 detected in the Philippines, PLoS One 8 (2013) e74221. [16] N. Khetsuriani, A. Lamonte-Fowlkes, S. Oberst, M.A. Pallansch, Centers for disease C, prevention enterovirus surveillance–United States, 1970–2005, MMWR Surveillance Summaries 55 (2006) 1–20. [17] Centers for Disease Control and Prevention, Clusters of acute respiratory illness associated with human enterovirus 68 – Asia, Europe, and United States, 2008–2010, MMWR Morb. Mortal Wkly. Rep. 60 (2011) 1301–1304. [18] C.M. Midgley, M.A. Jackson, R. Selvarangan, G. Turabelidze, E. Obringer, D. Johnson, et al., Severe respiratory illness associated with enterovirus D68 – Missouri and Illinois, 2014, MMWR Morb. Mortal Wkly. Rep. 63 (2014) 798–799. [19] K. Bragstad, K. Jakobsen, A.E. Rojahn, M.K. Skram, K. Vainio, M. Holberg-Petersen, et al., High frequency of enterovirus D68 in children hospitalised with respiratory illness in Norway, autumn 2014, Influenza Other Respir. Viruses 9 (2015) 59–63. [20] S. Esposito, A. Zampiero, L. Ruggiero, B. Madini, H. Niesters, N. Principi, Enterovirus D68- associated community-acquired pneumonia in children living in Milan, Italy, J. Clin. Virol. (2015) 94–96. [21] Biofire FilmArray Respiratory Panel. http://biofiredx.com/pdfs/FilmArray/ Info-Sheet-Respiratory-Panel-MRKT-PRT-0229-04.pdf. (accessed 24.04.15). [22] J. Zhuge, E. Vail, J.L. Bush, L. Singelakis, W. Huang, S.M. Nolan, et al., Evaluation of a real-time reverse transcription-PCR assay for detection of enterovirus D68 in clinical samples from an outbreak in New York state in 2014, J. Clin. Microbiol. 53 (2015) 1915–1920. [23] W.A. Nix, M.S. Oberste, M.A. Pallansch, Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens, J. Clin. Microbiol. 44 (2006) 2698–2704. [24] F. Hassan, A. Nguyen, A. Formanek, J.J. Bell, R. Selvarangan, Comparison of the BD Veritor system for Flu A + B with the Alere BinaxNOW influenza A&B card for detection of influenza A and B viruses in respiratory specimens from pediatric patients, J. Clin. Microbiol. 52 (2014) 906–910. [25] S.B. Selvaraju, R. Selvarangan, Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses, J. Clin. Microbiol. 48 (2010) 3870–3875. [26] P.A. Harris, R. Taylor, R. Thielke, J. Payne, N. Gonzalez, J.G. Conde, Research electronic data capture (REDCap)-a metadata-driven methodology and

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34] [35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

workflow process for providing translational research informatics support, J. Biomed. Inform. 42 (2009) 377–381. A. Waghmare, S.A. Pergam, K.R. Jerome, J.A. Englund, M. Boeckh, J. Kuypers, Clinical disease due to enterovirus D68 in adult hematologic malignancy patients and hematopoietic cell transplant recipients, Blood 125 (2015) 1724–1729. National Heart, Lung, Blood Institute. Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma. http://www.nhlbi.nih.gov/files/ docs/guidelines/asthgdln.pdf. Published 2007. Updated 2012 (accessed 24.04.15) 2008. J.M. Biagini Myers, J.M. Simmons, C.M. Kercsmar, L.J. Martin, V.V. Pilipenko, S.R. Austin, et al., Heterogeneity in asthma care in a statewide collaborative: the Ohio Pediatric Asthma Repository, Pediatrics 135 (2015) 271–279. S.L. Bratton, F.O. Odetola, J. McCollegan, M.D. Cabana, F.H. Levy, H.T. Keenan, Regional variation in ICU care for pediatric patients with asthma, J. Pediatr. 147 (2005) 355–361. J.S. Giuliano Jr., E.V. Faustino, S. Li, M.G. Pinto, M.F. Canarie, C.L. Carroll, et al., Corticosteroid therapy in critically ill pediatric asthmatic patients, Pediatr. Crit. Care Med. 14 (2013) 467–470. S.L. Bratton, C.J. Newth, A.F. Zuppa, F.W. Moler, K.L. Meert, R.A. Berg, et al., Critical care for pediatric asthma: wide care variability and challenges for study, Pediatr. Crit. Care Med. 13 (2012) 407–414. Centers for Disease Control and Prevention. Enterovirus D68 in the United States, 2014. www.cdc.gov/non-polio-enterovirus/outbreaks/EV-D68outbreaks.html. Published 2014. Updated January 8, 2015. (accessed 24.04.15). S.C. Yeung, G. Antonio, K.S. Ip, Flaccid paralysis of the limbs after an asthmatic attack, Pediatr. Neurol. 42 (2010) 133–136. J.I. Manson, Y.H. Thong, Immunological abnormalities in the syndrome of poliomyelitis-like illness associated with acute bronchial asthma (Hopkin’s syndrome), Arch. Dis. Child. 55 (1980) 26–32. J.C. Hayward, S.M. Gillespie, K.M. Kaplan, R. Packer, M. Pallansch, S. Plotkin, et al., Outbreak of poliomyelitis-like paralysis associated with enterovirus 71, Pediatr. Infect. Dis. J. 8 (1989) 611–616. K. Messacar, T.L. Schreiner, J.A. Maloney, A. Wallace, J. Ludke, M.S. Oberste, 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 385 (2015) 1662–1671. K.M. Wylie, T.N. Wylie, A. Orvedahl, R.S. Buller, B.N. Herter, V. Magrini, et al., Genome sequence of enterovirus D68 from St. Louis, Missouri, USA, Emerg. Infect. Dis. 21 (2015) 184–186. K.A. Poehling, K.M. Edwards, M.R. Griffin, P.G. Szilagyi, M.A. Staat, M.K. Iwane, et al., The burden of influenza in young children, 2004–2009, Pediatrics 131 (2013) 207–216. K.M. Edwards, Y. Zhu, M.R. Griffin, G.A. Weinberg, C.B. Hall, P.G. Szilagyi, et al., Burden of human metapneumovirus infection in young children, N. Engl. J. Med. 368 (2013) 633–643. K.L. Hon, W.S. Tang, T.F. Leung, K.L. Cheung, P.C. Ng, Outcome of children with life-threatening asthma necessitating pediatric intensive care, Ital. J. Pediatr. 36 (2010) 47. J. Pirie, P. Cox, D. Johnson, S. Schuh, Changes in treatment and outcomes of children receiving care in the intensive care unit for severe acute asthma, Pediatr. Emerg. Care 14 (1998) 104–108. K.M. James, T. Gebretsadik, G.J. Escobar, P. Wu, K.N. Carroll, S.X. Li, et al., Risk of childhood asthma following infant bronchiolitis during the respiratory syncytial virus season, J. Allergy Clin. Immunol. 132 (2013) 227–229.