key: cord-280859-3ff72mlq authors: Abe, Nozomi; Yasudo, Hiroki; Fukano, Reiji; Nakamura, Tamaki; Okada, Seigo; Wakiguchi, Hiroyuki; Okazaki, Fumiko; Shirabe, Komei; Toda, Shoichi; Okamoto, Reiko; Ouchi, Kazunobu; Ohga, Shouichi; Hasegawa, Shunji title: Multi‐season analyses of causative pathogens in children hospitalized with asthma exacerbation date: 2019-08-12 journal: Pediatr Allergy Immunol DOI: 10.1111/pai.13102 sha: doc_id: 280859 cord_uid: 3ff72mlq BACKGROUND: Respiratory viral and mycoplasma infections are associated with childhood asthma exacerbations. Here, we explored epidemiologic profile of causative pathogens and possible factors for exacerbation in a single center over a three‐year period. METHODS: Hospitalized asthmatic children with attack aged 6 months‐17 years were recruited between 2012 and 2015 (n = 216). Nasopharyngeal mucosa cell samples were collected from the participants and examined by reverse transcription‐polymerase chain reaction to detect rhinovirus (RV), respiratory syncytial virus (RSV), enterovirus (EV), parainfluenza virus (PIV), Mycoplasma pneumoniae, and others. Clinical features, laboratory data, asthma exacerbation intensity, and asthma severity were compared among participants. Epidemiologic profile of causative pathogens and possible factors for exacerbation were explored. RESULTS: Viruses and/or Mycoplasma pneumoniae were detected in 75% of the participants. Rhinovirus (48%) was the most commonly detected virus in the participants with single infection, followed by RSV (6%). The median age at admission in the RV group was significantly higher than that in the RSV group. Insufficient asthma control and allergen sensitization were significantly related to RV‐associated asthma exacerbation. There was no seasonality of pathogen types associated with asthma exacerbation although a sporadic prevalence of EV‐D68 was observehinovirud. Rhinovirus were repeatedly detected in multiple admission cases. CONCLUSION: Our three‐year analysis revealed that patients with RV infection were significantly prone to repeated RV infection in the subsequent exacerbation and good asthma control could prevent RV‐associated asthma development and exacerbation. Multiple‐year monitoring allowed us to comprehend the profile of virus‐ and/or mycoplasma‐induced asthma exacerbation. Respiratory viral infections are associated with the pathophysiology of childhood asthma exacerbation. 1 The major pathogens involved are human rhinovirus (RV), respiratory syncytial virus (RSV), enterovirus (EV), influenza virus, and human metapneumovirus (hMPV). 2 In previous studies, RSV has been associated with wheezing during infancy, whereas RV has been detected more frequently in school-age children with wheezing. 3 However, most studies on the association of viral infection with asthma exacerbation were conducted for only one season. [4] [5] [6] One season analysis is insufficient to comprehend the prevalence of virus-and mycoplasma-induced asthma exacerbation as viral or mycoplasma prevalence might show seasonal variation or some might be found temporary as an epidemic, suggesting that a longer-term analysis is needed. Therefore, this study aimed to clarify the association between asthma exacerbation and causative pathogens for three seasons. In addition, epidemiologic profile of causative pathogens and possible factors for exacerbation were explored. Asthmatic children hospitalized with asthma attack (n = 226) were enrolled at the Department of Pediatrics, Yamaguchi University Hospital, between April 2012 and May 2015. Five subjects were excluded due to lack of the consents from the patients and parents, and three subjects were excluded due to sufficient clinical and laboratory data. Two subjects were excluded because the nasopharyngeal samples were not properly obtained. As a result, 216 participants were enrolled for the analysis ( Figure S1 ). Bronchial asthma was diagnosed according to the Japanese Pediatric Guideline for the Treatment and Management of Bronchial Asthma 2012 (JPGL 2012), 7 which includes recurrent respiratory symptoms such as whistling, wheezing, difficulty in breathing, and chest tightness appearing during the night or early morning, chest auscultation for wheezing, respiratory function tests, IgE tests, and family and patients' past histories of allergic diseases. Asthma exacerbation was defined as respiratory symptoms such as wheezing, cough, difficulty in breathing, chest tightness with chest auscultation for wheezing, and/or decrease in airway flow relieved by inhaled short-acting beta-2 adrenergic agonists (SABA), and the intensity of asthma exacerbation was classified into four stages: mild, moderate, severe exacerbation, and respiratory failure according to JPGL 2012. 7 The level of asthma control was categorized in accordance with International Consensus on (ICON) Pediatric Asthma. 8 The criteria for hospitalization were episodes of difficulty in sleeping at night, or oral ingestion difficulty, and/or oxygen saturation of peripheral artery (SpO 2 ) < 95%. Patient demographic information was obtained from the hospital chart and questionnaires. Informed consent was obtained from the parents and/or the patients recruited in this study. This protocol was approved by the Institutional Review Board of Yamaguchi University Hospital (H24-11). Nasopharyngeal samples were collected from all patients. Sampling from the nasopharyngeal and transportation of the specimen to Yamaguchi Prefectural Institute of Public Health and Environment were performed by well-experienced pediatricians. The samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) to amplify specific genes from EV, 9 RSV, 10 PIV, 11 hMPV, 12 RV (subtypes A, B, and C), 13 influenza viruses (subtypes A and B), adenoviruses (AdV), 14 Coxsackie virus (Cox), 9 human coronavirus (HCoV), 15 and Mycoplasma pneumoniae 16 Pathogen-specific clinical and laboratory features such as bronchial asthma severity, intensity of asthma exacerbation, the level of the asthma control, and hematologic and immunologic tests were analyzed both by univariate analyses (Mann-Whitney U test or • Asthma children (n = 216; 6 months-17 years) hospitalized with exacerbation were enrolled consecutively for 3 years in a single center. • Multi-season analyses allowed us to explore epidemiological profile of causative pathogens and possible factors for exacerbation. • Rhinovirus (RV) and respiratory syncytial virus were most frequently detected. • Although a sporadic epidemic of EV-D68 was detected, no seasonality in pathogen types were observed. • Children with RV-associated exacerbation were more likely infected with RV on the subsequent admission. • Multivariate analyses suggested that good asthma control and avoidance of allergy sensitization could prevent RV-associated asthma. chi-square test) and by multiple logistic regression analysis. Analyses and calculations were performed using BellCurve for Excel and StatFlex for Windows Ver 7.0 (Artech Inc). The characteristics of clinical and laboratorial data were summarized ( 48%) was the most commonly detected virus in participants with asthma exacerbation followed by RSV (n = 10; 6%). As RV and RSV were mostly detected in the participants with asthma exacerbation, we compared clinical characteristics among participants with single infection in whom we detected RV or RSV ( Table 1 ). The median age and serum house-dust mite IgE levels at admission were significantly higher in the RV group than those in the RSV groups (P < .01). There was no significant difference in body temperature, SpO 2 , duration of admission, the median number of peripheral white blood cells (WBCs), and serum levels of C-reactive protein (CRP) between the two groups. No significant difference was observed in the intensity of asthma exacerbation on admission, bronchial asthma severity, and the level of asthma control between the groups as well. Although RV type A and type C were detected in the majority of participants infected with RV (type A, n = 33; type B, n = 1; type C, n = 52; unknown, n = 1), there were no significant differences in TA B L E 1 Clinical characteristics, asthma exacerbation intensity, and asthma severity in the total subjects and the asthmatic subjects with single infection of RV and RSV body temperature, SpO 2 , duration of admission, laboratory data, intensity of asthma exacerbation, bronchial asthma severity, and the level of asthma control between the two groups (data not shown). To explore epidemiologic features of RV-associated asthma exacerbation, multiple logistic regression analyses were performed by setting the status of RV vs. non-RV-associated exacerbation as objective variable (Table S1 ). It revealed that insufficient control status and higher serum house-dust mite IgE levels are related to RV-associated asthma exacerbation requiring hospitalization (P < .05 and P < .01, respectively). Next, clinical characteristics, intensity of asthma exacerbation, bronchial asthma severity, and the level of asthma control were compared in the asthmatic patients with single infection and superinfection ( Table 2 ). The median age and house-dust mite IgE levels at admission were significantly higher in the single infection group than those in the superinfection group (P < .001), whereas there were no significant differences in body temperature, SpO 2 , duration of hospitalization, serum levels of CRP, intensity of asthma exacerbation, and bronchial asthma severity between the two groups. Multiple logistic analyses revealed that elder age and higher housedust mite IgE levels at admission were related to single infection as well (P < .001 and P < .05, respectively, data not shown). Seasonal pathogen distribution associated with asthma exacerbation has not been conducted so far because most studies on the association of pathogens with asthma exacerbation were conducted for only one season. 4 distribution based on the month of admission ( Figure 2 ). There were no seasonal peaks in virus and/or M pneumoniae detections, although RV and PIV were detected in all seasons. Next, we evaluated the detected pathogens in the patients with asthma exacerbation on multiple admissions. Among the 31 patients on multiple admissions, RVs were repeatedly detected in 18 patients on subsequent admissions as well as on the initial admission (Table 3) , which showed that those with initial RV infection associated asthma exacerbation were likely to be infected with RV on the subsequent admission as well (P < .01). In this study, six asthmatic patients had EV infection, and EV-D68 was detected in all six patients. We evaluated the relationship between EV-D68 infection and hospital admission due to asthma exacerbation ( Figure 3) . A low prevalence was observed only in 2013, but not in 2012 or 2014, suggesting that EV-D68 prevalence was not annual, unlike that of RV, RSV, or PIV. In this study, RV (48%) was the most detected virus among subjects with asthma exacerbation followed by RSV (6%), and the age on admission was significantly higher in the RV group than in the RSV group. The detection frequency of RV and RSV in our study was consistent with that in previous reports on the relationship between causative viruses and exacerbation in asthmatic patients, suggesting that our study illustrates the actual virus prevalence associated with asthma exacerbation. 2, 3 Heymann et al 20 reported prevalence of rhinovirus infection was much less in the wheezing patients aged <3 years compared to those above the age. Thus, we compared RV prevalence between the patients with <2 years old (n = 67) and those above the age (n = 149). Pathogens were identically detected in 79% in both groups. However, RV was detected in only 38% of the patients <2 years of age, whereas the virus was detected in 57.6% of those ≥2 years. This difference in the RV detection rate was statistically significant (P = .002), which explains an overall lower prevalence of RV in our study (data not shown). There are several studies showing that RV infection is more associated with wheezing in older children. 3, 21 In our three-season analysis, the median age of subjects with RV infection was significantly higher than that of participants with RSV infection, which was consistent with the results of previous reports based on one season analysis. Allergic sensitization was reported to be related to RV-associated wheeze. 22 Interestingly, we observed a closer association of house-dust mite-specific IgE with the RV group than with the RSV group. Multiple logistic regression analysis revealed that there is a closer association of insufficient asthma control and house-dust mite-specific IgE with the RV group than with the non-RV group. These suggest that RV-associated asthma exacerbation is related to allergen sensitization and that good asthma control could prevent RV-associated asthma exacerbation. Rhinovirus type C is reported to cause more severe attacks than other RV types. 23, 24 However, there was no significant difference in the intensity of asthma exacerbation between the RV type A and type C groups in our study. Zheng et al 25 reported that RV type A infection with high viral load leads to severe asthma exacerbation; we therefore speculate that the difference in the viral load of subjects between RV type A and type C may reflect similar results in asthma exacerbation intensity and asthma severity in our study. There was no significant difference in intensity of asthma exacerbation and bronchial asthma severity between the single infection group and the superinfection group, suggesting that superinfection does not exert additional effect on asthma exacerbation. Interestingly, we observed the association of elder age and higher house-dust mite IgE levels with single infection in multiple logistic analyses. Allergic sensitization was suggested as leading to increased susceptibility to RV, and RV-associated wheeze. 22 Our results confirmed the reported finding of exacerbation of asthma associated with RV. It is interesting that RVs were repeatedly detected in multiple admission cases based on our three-season analysis; that is, patients with initial RV-associated asthma exacerbation were likely to be in- was only detected in 2013 during the three-season study, which is in agreement with the outbreak that occurred in Japan during the study period. 27 We previously reported EV-D68 outbreak in the same area in 2010. 28 There has never been an outbreak of EV-D68 since autumn 2010 to spring 2015 (data not shown). This suggests that EV-D68 outbreak is not an annual but a time-limited event. There are some limitations in this study. First, this is a pilot study conducted in a single center. Further multi-center studies involving different regions are warranted to confirm the relationship between causative pathogens and asthma exacerbation. Second, the patients with asthma exacerbations requiring hospitalization were recruited in our study, which may have resulted in a selection bias; that is, children presenting with mild exacerbation in relatively good control were not included. Therefore, there can be some limitation in analyzing the impact of pathogens for the entire range of asthma exacerbation. In conclusion, our three-season analysis on the prevalence of causative pathogens in hospitalized children with asthma exacerbation revealed that RV and RSV were most frequently detected. Insufficient asthma control and allergen sensitization were clearly related to RV-associated asthma exacerbation. Rhinovirus were repeatedly detected in multiple admission cases in our three-season analysis, suggesting that repetitive RV infections could contribute to subsequent aggravation of asthma. Putting these findings together, good asthma control could prevent RV-associated asthma development and exacerbation. Additionally, epidemic of EV-D68 infection was observed during a few months, suggesting the requirement for multiple-year monitoring to comprehend the prevalence of asthma exacerbation with viral infections including temporary small epidemic. We are grateful to Prof. Kiyoshi Ichihara of our university, a biostatistician with medical background, for his dedicated support for the statistical analyses and writing to improve the scientific quality of our manuscript. There are no conflicts of interest to declare. NA and HY contributed equally to this work. HY, KS, KO, SO, and SH were the principal investigators taking primary responsibility for the manuscript. RF and SO performed the clinical management with helpful discussion for the completion of the study. NA, TN, HW, FO, ST, and RO took responsibility for the diagnosis and data collection. The nucleotide sequences determined in this study were deposited in the DDBJ under the accession numbers AB618506-AB618528. Role of viral infections in the development and exacerbation of asthma in children Regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review Rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. IgE and eosinophil analyses Community study of role of viral infections in exacerbations of asthma in 9-11 year old children Prevalence of viral respiratory tract infections in children with asthma Distribution and seasonality of rhinovirus and other respiratory viruses in a crosssection of asthmatic children in Trinidad, West Indies Japanese pediatric guideline for the treatment and management of bronchial asthma 2012 International consensus on (ICON) pediatric asthma Molecular diagnosis of human enteroviruses by phylogeny-based classification using the VP4 sequence Reliable detection of respiratory syncytial virus infection in children for adequate hospital infection control management Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR Human metapneumovirus infection in febrile children with lower respiratory diseases in primary care settings in Hiroshima Human rhinovirus 87 identified as human enterovirus 68 by VP4-based molecular diagnosis Pring-Akerblom P. Rapid and quantitative detection of human adenovirus DNA by real-time PCR Frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction Chlamydophila pneumoniae, Mycoplasma pneumoniae infections, and asthma control Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens Surveillance of macrolide-resistant Mycoplasma pneumonia in Beijing Macrolide-resistant Mycoplasma pneumoniae infection Viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing Prevalence of respiratory viral infection in children hospitalized for acute lower respiratory tract diseases, and association of rhinovirus and influenza virus with asthma exacerbations Rhinovirus and preschool wheeze Patient characteristics and severity of human rhinovirus infections in children Human rhinovirus C: age, season, and lower respiratory illness over the past 3 decades Epidemiological analysis and follow-up of human rhinovirus infection in children with asthma exacerbation Global emergence of enterovirus D68: a systematic review Spike" in acute asthma exacerbations during enterovirus D68 epidemic in Japan: a nationwide survey Enterovirus 68 infection in children with asthma attacks: virusinduced asthma in Japanese children Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article Multi-season analyses of causative pathogens in children hospitalized with asthma exacerbation