key: cord-0917118-coqvurgm authors: Zuccotti, Gianvincenzo; Dilillo, Dario; Zappa, Alessandra; Galli, Erica; Amendola, Antonella; Martinelli, Marianna; Pariani, Elena; Salvini, Filippo; Tanzi, Elisabetta; Riva, Enrica; Giovannini, Marcello title: Epidemiological and clinical features of respiratory viral infections in hospitalized children during the circulation of influenza virus A(H1N1) 2009 date: 2011-05-25 journal: Influenza Other Respir Viruses DOI: 10.1111/j.1750-2659.2011.00264.x sha: 46dd3216b2b02daaa3cfa9fe0c6108444fdfae1e doc_id: 917118 cord_uid: coqvurgm Please cite this paper as: Zuccotti et al. (2011) Epidemiological and clinical features of respiratory viral infections in hospitalized children during the circulation of influenza virus A(H1N1) 2009. Influenza and Other Respiratory Viruses 5(6), e528–e534. Background Seasonal influenza viruses and respiratory syncytial virus (RSV) are primary causes of acute respiratory tract infections (ARTIs) in children. New respiratory viruses including human metapneumovirus (hMPV), human bocavirus (hBoV), and influenza 2009 A(H1N1) virus have a strong impact on the pediatric population. Objectives To evaluate epidemiological and clinical features of ARTIs in hospitalized children. Methods From December 1, 2008, to December 31, 2009, all children under age fifteen (n = 575) hospitalized for ARTIs were investigated for influenza A (subtype H1N1, H3N2, and 2009 H1N1) and B, RSV A and B, hMPV, and hBoV by PCR. Results Fifty‐one percent of samples were positive for these respiratory viruses. The frequencies of virus detection were RSV 34·1%, hBoV 6·8%, hMPV 5%, seasonal influenza A 5%, and seasonal influenza B 0%. From April 2009, 11·6% of collected samples were influenza 2009 A(H1N1) positive. Respiratory syncytial virus activity peaked in January, hBoV in February, and hMPV in April. Seasonal influenza A was detected only between January and April 2009, while influenza 2009 A(H1N1) peaked in November. Respiratory syncytial virus and hMPV were mainly associated with lower respiratory tract infections (LRTIs) and with necessity of O(2) administration. The 2009 pandemic influenza was more frequently detected in elder children (P < 0·001) and was associated with higher, longer‐lasting fevers compared with other viral infections (P < 0·05). Conclusions All considered viruses were involved in LRTIs. The primary clinical relevance of RSV and a similar involvement of both seasonal influenza and emerging viruses investigated were observed on the pediatric population. Acute respiratory tract infections (ARTIs) are associated with significant morbidity worldwide; viruses are by far the most common causes of ARTIs, especially among young children. 1 In particular, seasonal influenza viruses (type A and B) and respiratory syncytial virus (RSV) are the main etiological agents during the epidemic period (between October and April in the northern hemisphere). 2,3 RSV infection is usually much more frequently identified than influenza in children; indeed, about 77% of infants have had an RSV infection before 5 years of age. This virus is the main cause of bronchiolitis, which is one of the major reasons of hospitalization in children under 2 years of age. 4 However, recent data show the important clinical role of seasonal influenza in children. In fact, the rate of hospitalization for seasonal influenza is not <3AE6 per 10 000 child ⁄ year, and hospitalization can also regard previously healthy children and children older than 2 years. 4, 5 The recent epidemiological scenario has been enlivened by the identification and emergence of several pathogens with an airborne transmission pathway such as human metapneumovirus (hMPV), first isolated in 2001, 6 human bocavirus (hBoV), discovered in 2005, 7 and influenza virus 2009 A(H1N1), identified in 2009, and responsible for the first pandemic of the new millennium. 8 HMPV and hBoV are isolated in 3AE9-16% of children hospitalized for ARTIs. The first is associated with a large spectrum of clinical manifestations that range from mild upper respiratory tract disease to severe bronchiolitis and pneumonia, 9, 10 and thus, it is considered to be one of the most important respiratory emerging viruses, while the second seems to have a marginal role as it causes mainly upper respiratory tract infections (URTIs), when detected alone. 11 The 2009 pandemic influenza affected the pediatric population in 60% of cases 8 causing a significant number of recovery also in children aged over 5 years. It seems to cause generally mild disease, similar to those of seasonal influenza 12 , while severe manifestations seem concentrated in patients with risk factors. This study aimed to evaluate the frequency and the demographic and clinical features of ARTIs caused by known viruses (i.e., RSV and seasonal influenza) and newly identified viruses (i.e., influenza 2009 A(H1N1), hMPV, and hBoV) in children hospitalized for ARTI in Milan (Italy) from December 2008 to December 2009. From December 1, 2008, to December 31, 2009, we enrolled 575 children aged between 0 and 15 years hospitalized for an ARTI in two Pediatric Clinics at the University of Milan ('L. Sacco' and 'S. Paolo' Hospitals). After informed consent was obtained from the parents, an oropharyngeal swab (Plain Swabs; Copan, Brescia, Italy) was collected from each child within 24 hour after hospital admission and tested at the Department of Public Health-Microbiology-Virology, University of Milan, using PCR assays to detect viral respiratory pathogens (i.e., influenza type A, subtype H1N1, H3N2, and 2009 H1N1; influenza type B; RSV type A and B; hMPV; and hBoV). A standardized datasheet was used to record sociodemographic data (age, gender, risk factors) obtained through parents interview and clinical information (duration of hospitalization, detailed disease signs and symptoms before and during hospitalization, prescribed drug therapy) by medical chart abstraction. For this study, children <15 years of age hospitalized with symptoms of ARTI such as cough, rhinitis, sore throat, wheezing, panting, dyspnea, or apnea were enrolled. Patients were assessed and categorized according to diagnosis of upper respiratory tract infections, wheezy bronchitis, bronchitis, bronchiolitis, and pneumonia on the basis of clinical and roentgenographic findings. The criteria proposed by Ruuskanen and Ogra 13 were used for definitions of bronchiolitis, pneumonia, and wheezy bronchitis. Acute illnesses indicated as upper respiratory infections (URTIs) were characterized by cough, rhinorrhea, sore throat, and ⁄ or otitis media, with normal thoracic objectivity and X-ray. An acute illness characterized by cough, rhonchi, and diffuse expiratory wheezing upon thoracic auscultation was diagnosed as wheezy bronchitis, while a illness with the same characteristic but without expiratory wheezing in any phase of its course was diagnosed as bronchitis. Bronchiolitis was diagnosed when dyspnea, tachypnea, diffuse small crackles upon thoracic auscultation, and roentgenographic evidence of hyperinflation of the lung with or without areas of collapse were present in children younger than 2 years of age. Pneumonia diagnosis was based on auscultation of pathological breath sounds, such as small crackles or decrease ⁄ absence of vesicular sound, in a zone of the chest, and on radiographic findings of lung parenchymal involvement with interstitial-alveolar infiltrates and ⁄ or consolidation. The term lower respiratory tract infections (LRTIs) was used to indicate acute illness with the presence of signs of lower airway involvement (tachypnea, dyspnea, wheezing, rhonchi, or rales) and ⁄ or a positive chest X-ray, so it includes bronchitis, wheezy bronchitis, bronchiolitis, and pneumonia. Nucleic acid extraction was conducted using a commercial method (NucliSENS Ò , miniMAG Ò ; Biomérieux, Marcy L'Etoile, France). For RNA virus detection, cDNA was synthesized with pd(N)6 random hexamer primers (Amersham Biosciences, Little Chalfont, UK) using an MMLV reverse transcriptase (Invitrogen Tech-Line, Carlsbad, CA, USA). Viral detection was performed by PCR assays. To simultaneously detect and type seasonal A and B influenza viruses, a one-step real-time RT-multiplex-PCR assay was performed using primer ⁄ probe sets for two different genome regions: the matrix region of influenza type A virus and the nucleoprotein region of influenza type B virus. 14 Influenza A-positive samples were subtyped using an RT-multiplex-PCR assay with specific primers for the hemagglutinin gene of influenza A ⁄ H1 and A ⁄ H3 viruses. 15 Pandemic 2009 A(H1N1) influenza virus was detected using a onestep real-time RT-PCR, in accordance with the Centers for Disease Control and Prevention guidelines. 16 RSV A and RSV B were identified by multiplex nested PCR (fusion gene, 336 and 582 bp, respectively) using specific primer sets. 17 Two nested PCR assays were performed to detect a 151-bp (nt. 44-195) fragment of the matrix gene of hMPV and a 354-bp (nt. 2351-2704) fragment of the nucleoprotein gene of hBoV, 18 respectively. Appropriate positive and negative controls were included in any PCR assay. Data were expressed as median (interquartile range, IQR) and percentages (95% confidence intervals, 95% CI) as appropriate. Comparisons between groups were performed using the chi-square test or Fisher's exact test. A P-value < 0AE05 was considered statistically significant (twotailed test). All statistical analyses were performed using OpenEPI software, version 2.2.1. 19 From December 2008 to December 2009, 575 children hospitalized for ARTIs (338 boys and 237 girls; median age 9AE0 months, IQR 3AE0-24AE0 months) were enrolled. Twentyfour children were not enrolled because guardian consent was lacking. Children were divided into five age-groups: <6 months (n = 242; 42AE1%, 95% CI: 38AE1-46AE2), 6-11 months (n = 77; 13AE4%, 95% CI: 10AE8-16AE4), 12-23 months (n = 106; 18AE4%, 95% CI: 15AE4-21AE8), 2-5 years (n = 99; 17AE2%, 95% CI: 14AE3-20AE5), and 6-15 years (n = 51; 8AE9%, 95% CI: 6AE7-11AE4). Patients were more frequently affected by LRTIs than URTIs (67AE8%, 95% CI: 63AE9-71AE5 versus 32AE2%, 95% CI: 28AE4-36AE1; P < 0AE0001). Bronchiolitis was diagnosed in 145 children (25AE2%, 95% CI: 21AE8-28AE9), pneumonia in 151 (26AE3%, 95% CI: 22AE8-30AE0), wheezy bronchitis in 57 (9AE9%, 95% CI: 7AE7-12AE6), and bronchitis in 37 (6AE4%, 95% CI: 4AE6-8AE7). Molecular investigations revealed viral gene sequences in 293 (51AE0%, 95% CI: 46AE9-55AE0) samples. Respiratory syncytial virus was detected in 196 (34AE1%, 95% CI: 30AE3-38AE0) of collected samples. Of these, 67 (34AE2%, 95% CI: 27AE8-41AE0) were RSV A positive and 129 (65AE8%, 95% CI: 59AE0-72AE2) were RSV B positive (P < 0AE05). HBoV was detected in 39 (6AE8%, 95% CI: 5AE0-9AE1) and hMPV and seasonal influenza A in 29 (5AE0%, 95% CI: 3AE5-7AE1) samples. Regarding seasonal influenza A-positive samples, 25 (86AE2%, 95% CI: 70AE0-95AE5) were subtype H3 and 4 (13AE8%, 95% CI: 4AE5-30AE0) were subtype H1 (P < 0AE05 Mixed infections were detected in 8AE2% of ARTIs (Table 1) , and in particular, in 41AE0% of samples positive for hBoV, 9AE2% of samples positive for RSV, and 7AE7% of samples positive for influenza 2009 A(H1N1) (hBoV coinfections versus RSV and influenza 2009 A(H1N1); P < 0AE003). Single and co-infections were associated with LRTIs with similar percentage (83AE3% versus 78AE8%, P = 0AE3). Viral infections were detected more frequently in children younger than 24 months of age (84AE3% versus 15AE7%, P < 0AE05). Respiratory syncytial virus (type A and B) accounted for 59AE5% of infections in children younger than 6 months, while elder children experienced seasonal influenza, hBoV, RSV, and hMPV infections with similar frequencies ( Figure 2) . The median age of children with a 2009 A(H1N1) influenza was significantly higher than that of children infected with the other viruses (median age: RSV: 3 months, For analysis of the demographic and clinical features of children with viral respiratory infection, co-infected patients were excluded. Respiratory syncytial virus A and B were considered together because their clinical and sociodemographic features did not differ significantly (P > 0AE05). Fever was present in all patients, except approximately one-third of RSV-infected children. The average duration of fever was significantly longer in children with 2009 pandemic influenza than in RSV-positive or hMPV-positive children (4AE9 versus 3AE6 and 2AE8 days, respectively, P < 0AE05); 70AE8% of children with 2009 pandemic influenza had high fever ( ‡39°C) ( Table 2) . Cough and rhinitis were less associated with seasonal influenza than with other infections (Table 2) . Dyspnea, hypoxia, and the resulting necessity of O 2 administration were significantly associated with RSV (P < 0AE05). The 2009 A(H1N1) influenza-positive and hMPV-positive children had feeding difficulties compared with children with the other infections (hMPV: 100%; influenza 2009 A(H1N1): 87AE5%; RSV: 68AE0%; hBoV: 56AE5%; seasonal influenza A: 50AE0%; P < 0AE05) and received antibiotics more frequently than RSV-positive children (hMPV: 100%; influenza 2009 A(H1N1): 100%; RSV: 79AE8%; P < 0AE05). No patients required intensive care ( Table 2) . All viruses considered in this study were involved in LRTIs with different frequencies. Lower respiratory tract infections were associated with 91AE6% of RSV, 66AE6% of hMPV, 56AE5% of hBoV, 50AE0% of seasonal influenza A, and 41AE7% of 2009 A(H1N1) influenza infections (RSV-LRTI versus other viruses-LRTI, P < 0AE05), ( Table 2 ). In particular, bronchiolitis was more frequently associated with RSV than with hMPV or hBoV infections (55AE6% bronchiolitis-RSV versus 20% bronchiolitis-hMPV and 8AE7% bronchiolitis-hBoV, P < 0AE05). No cases of bronchiolitis were observed in association with seasonal or 2009 pandemic influenza. RSV-positive and hMPV-positive patients required use of bronchodilators more frequently than children infected by the other viruses (RSV: 84AE8%; hMPV: 79AE2%; hBoV: 43AE5%; influenza A: 35AE0%; 2009 A(H1N1) influenza: 37AE5%; P < 0AE05) ( Table 2) . No influenza antivirals were administered to any patient during the study period. The main risk factors for ARTIs observed in patients with a viral infection were exposure to environmental tobacco smoke (25AE3%), family history of atopic disease (23AE9%), and the presence of at least one sibling (15AE3%) ( Table 2 ). This report describes the frequencies, the demographic features, and the clinical features of viral infections identified following surveillance for ARTIs in children hospitalized for ARTI in Milan (Italy) from December 2008 to December 2009. Fifty-one percent of children hospitalized for ARTIs had an infection caused by either known viruses (i.e., RSV and seasonal influenza) or newly identified viruses (i.e., 2009 A(H1N1) influenza, hMPV, and hBoV). Probably, most of samples that resulted negative for the considered viruses could have been positive for other viruses not included in this study, such as parainfluenza viruses, coronaviruses, adenoviruses, and rhinoviruses. The data revealed that children younger than 2 years of age, particularly boys, were at high risk of hospitalization for ARTIs compared with older patients. 20, 21 Respiratory syncytial virus was the main agent associated with ARTI in children, especially in patients younger than 6 months, and was responsible for almost all LRTIs. The primary clinical relevance of RSV in children is well characterized. Serologic evidence indicates that nearly all children have been infected by RSV within the first 2 years of life. 22 Hospitalization is required in approximately 0AE5-2% of cases and 80% of these occur in the first year of life. 23 In Europe, RSV accounts for 42-45% of hospital admission for LRTIs in children younger than 2 years; in particular, in studies on hospitalized children, RSV is associated with bronchiolitis in 60-90% of cases and with pneumonia in 25-50% of cases. 24 No vaccines against this virus are available so far, but prophylaxis with palivizumab (a human, 25 The two most important studies on RSV epidemiology in Italy have shown that the epidemic starts in October-November and ends in April-May, with peak incidence in February. 3, [26] [27] [28] [29] In this study, RSV activity peaked in January; however, in contrast with previous studies, it rose gradually from October 2009. Seasonal influenza viruses were identified in 5% of samples and between December 2008 and April 2009 only. No influenza B viruses were detected during the study period. The absence of seasonal influenza viruses at the end of 2009 in our study is in agreement with data from both the Italian Influenza Surveillance Network (INFLUNET) 30 and the European Centre for Disease Prevention and Control. 31 In this study, hMPV was detected in 5% of patients. These data are in agreement with published literature, in which frequencies of hMPV infections ranged from 3AE9% to 16%. Some authors have claimed a clinical similarity between hMPV and RSV infections. 32, 33 In the population analyzed, hMPV was associated with LRTIs, dyspnea, and hypoxia less frequently than RSV. HBoV infection was involved in 6% of ARTIs and in 41% of all co-infections identified. These data are in agreement with studies investigating hBoV infection in ARTIs, which have found a prevalence of 1AE5-18AE3%, 11,34 with a 42AE5% mean percentage of co-infections with other viral pathogens. 34 In most cases, hBoV had a marginal clinical importance when identified alone, causing an URTI in 43AE5% and requiring oxygen in 8AE7% of cases. However, according to other studies, this study raises the possibility that hBoV is also associated with severe ARTIs such as bronchiolitis, 35, 36 asthma exacerbations, 37,38 and pneumonia. 39 The seasonal peak of hBoV varies among studies, but is usually described in the early winter. 34 In this study, hBoV peak was observed in February, and its circulation was low at the end of 2009. In agreement with data registered in European countries, 30, 31, 40 the present study demonstrates that the 2009 pandemic influenza virus peaked in November 2009; In agreement with published literature, 41, 42 our data indicate that children with high hospitalization risk for pandemic influenza were not only those younger than 2 years of age or with chronic disease (as usually observed for seasonal influenza), but also children older than 5 years of age, accounting for more of 25% of recovery from 2009 pandemic influenza. Finally, 2009 pandemic influenza caused a clinical manifestation similar to seasonal influenza with mild severity, although these children had bad general conditions because of fever. In conclusion, all viruses considered in this study circulated in Italy and were involved in LRTIs in children. These findings confirm the primary clinical relevance of RSV, and a similar involvement of both the seasonal influenza and the emerging viruses investigated in ARTIs among hospitalized children. National disease burden of respiratory viruses detected in children by polymerase chain reaction The global state of influenza in children Prevalence of respiratory syncytial virus infection in Italian infants hospitalized for acute lower respiratory tract infections, and association between respiratory syncytial virus infection risk factors and disease severity Incidence, complications and risk factors for prolonged stay in children hospitalized with community-acquired influenza Multistate surveillance for laboratory-confirmed, influenza-associated hospitalizations in children A newly discovered human pneumovirus isolated from young children with respiratory tract disease Human bocavirus: passenge or pathogen in acute respiratory tract infections? Emergence of novel swine-origin influenza A (H1N1) virus in humans Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience Impact of human bocavirus on children and their families Update on the 2009 pandemic influenza A H1N1 in children Respiratory syncytial virus Performance testing of two new one-step real time PCR assay for detection of human influenza and avian influenza viruses isolated in humans and respiratory syncytial virus Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses CDC protocol of realtime RT-PCR for influenza A (H1N1) Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay Epidemiological profile and clinical associations of human bocavirus and other human parvoviruses OpenEpi: Open Source Epidemiologic Statistics for Public Health Occurence of respiratory virus: time, place and person Hospital admission in children up to the age of 2 years Occurrence of groups A and B RSV over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children Epidemiology of RSV in Washington, DC. I. Importance in different respiratory tract disease syndromes and temporal distribution Impact of severe disease caused by respiratory syncytial virus in children living in developed countries Preventing RSV bronchiolitis in vulnerable infants: the role of palivizumab Respiratory syncytial virus activity-United States Respiratory syncytial virus epidemics: the ups and downs of a seasonal virus Osservatorio VRS'' Study Group. Incidence of respiratory syncytial virus infection in infants and young children reffered to the emergency departements for lower respiratory tract disease in Italy Four year incidence of respiratory syncytial virus infection in infants and young children referred to emergency departments for lower respiratory tract diseases in Italy: the ''Osservatorio VRS'' Study Available at http: ⁄ ⁄ www.salute. gov.it ⁄ influenza ⁄ documenti ⁄ virologia ⁄ AggVir23_10.pdf (Accessed European Centre for Disease Prevention and Control. Weekly influenza surveillance overview Human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections Human bocavirus in French children The human bocaviruses: a review and discussion of their role in infection Human bocavirus in Italian patients with respiratory disease André oletti L. Human bocavirus quantitative DNA detection in French children hospitalized for acute bronchiolitis Human bocavirus and acute wheezing in children Human bocavirus in children with acute asthma Human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in Thailand Impact of the 2009 influenza A(H1N1) pandemic wave on the pattern of hibernal respiratory virus epidemics Risk factors and outcomes among children admitted to hospital with pandemic H1N1 influenza and composition of the 2010-11 influenza vaccine Asthma, atopic dermatitis, allergic rhinitis, or anaphylaxis in first-degree relatives.