Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.     

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.     

254例急性呼吸道感染病例病原学调查分析

目的 分析急性呼吸道感染病例的病毒病原构成,掌握主要病原体活动水平及变化规律,了解丰台地区急性呼吸道感染病原谱.方法 以2010-2012年成人急性呼吸道感染病例为研究对象,收集病例基础信息,采集鼻咽拭子标本,使用多重PCR及RT-PCR对流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、偏肺病毒、冠状病毒、鼻病毒、博卡病毒和肺炎支原体等9种病原进行检测,数据分析使用SPSS 17.0软件.结果 254例标本中154例检测阳性,阳性率为60.63％,男女阳性检出率无统计学差异.冬春季致病病原体以流感病毒、副流感病毒及支原体为主,夏季仅检出副流感和支原体.流感样病例的主要病原体为流感病毒及副流感病毒;肺炎病例致病病原体主要为肺炎支原体.2010-2011年流感流行季甲型流感病毒为优势病原,2011-2012年流感流行季乙型流感病毒为优势病原.结论 2010-2012年间,北京丰台地区引起成人急性呼吸道感染的主要病原为流感病毒、支原体和副流感病毒.不同季节的优势病原不同.     

Contribution of common and recently described respiratory viruses to annual hospitalizations in children in South Africa

The contribution of viruses to lower respiratory tract disease in sub-Saharan Africa where human immunodeficiency virus may exacerbate respiratory infections is not well defined. No data exist on some of these viruses for Southern Africa. Comprehensive molecular screening may define the role of these viruses as single and co-infections in a population with a high HIV-AIDS burden. To address this, children less than 5 years of age with respiratory infections from 3 public sector hospitals, Pretoria South Africa were screened for 14 respiratory viruses, by PCR over 2 years. Healthy control children from the same region were included. Rhinovirus was identified in 33% of patients, RSV (30.1%), PIV-3 (7.8%), hBoV (6.1%), adenovirus (5.7%), hMPV (4.8%), influenza A (3.4%), coronavirus NL63 (2.1%), and OC43 (1.8%). PIV-1, PIV-2, CoV-229E, -HKU1, and influenza B occurred in <1.5% of patients. Most cases with adenovirus, influenza A, hMPV, hBoV, coronaviruses, and WU virus occurred as co-infections while RSV, PIV-3, and rhinovirus were identified most frequently as the only respiratory pathogen. Rhinovirus but not RSV or PIV-3 was also frequently identified in healthy controls. A higher HIV sero-prevalence was noticed in patients with co-infections although co-infections were not associated with more severe disease. RSV, hPMV, PIV-3, and influenza viruses had defined seasons while rhinovirus, adenovirus, and coronavirus infections occurred year round in this temporal region of sub-Saharan Africa.     

Prevalence of human metapneumovirus among hospitalized children younger than 1 year in Catalonia, Spain

Human metapneumovirus was discovered recently respiratory virus implicated in both upper and lower respiratory tract infection. In children, the clinical symptoms of human metapneumovirus are similar to those produced by respiratory syncytial virus, ranging from mild to severe diseases such as bronchiolitis and pneumonia. The aim of the present study was to describe the prevalence of human metapneumovirus and other common respiratory viruses among admitted to hospital infants. From January 2006 to June 2006, 99 nasopharyngeal aspirates were collected from hospitalized children younger than 12 months in order to study respiratory viruses. Human metapneumovirus detection was performed by cell culture and two RT-PCR targeting on polymerase and fusion genes. The latter gene was used for phylogenetic analysis. In 67/99 children (67%) at least one viral pathogen was identified, the viruses detected most frequently were respiratory syncytial virus (35%), human metapneumovirus (25%) and rhinovirus (19%). The results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among children less than 12 months; (2) children infected with human metapneumovirus were significantly older than those infected by respiratory syncytial virus; (3) human metapneumovirus was associated more frequently with pneumonia whereas respiratory syncytial virus was only detected in patients with bronchiolitis; (4) there was a clear epidemiological succession pattern with only a small overlap among the viruses detected most frequently; (5) all human metapneumovirus samples were clustered within sublineage A2.     

Newly identified respiratory viruses associated with acute lower respiratory tract infections in children in Lanzou,China, from 2006 to 2009

Nasopharyngeal aspirates were collected from 813 children <14 years old with acute lower respiratory tract infections in Lanzhou, China, from December 2006 to November 2009. PCR or RT-PCR was used to screen for the presence of 10 respiratory viruses. Viral agents were identified in 73.92% (601/813) of specimens, including RSV in 40.71%, hMPV in 6.15%, IFVA in 7.13%, IFVB in 0.98%, PIV1-3 in 7.87%, HCoV-HKU1 in 2.21%, HCoV-NL63 in 3.81%, HRV in 19.93%, AdV in 7.50% and HBoV in 11.56%. Two or more viruses were detected in 34.44% (280/813) of cases. The newly identified respiratory viruses, HBoV, hMPV, HCoV-HKU1 and HCoV-NL63, accounted for 22.01% of the detected viral pathogens. RSV and HRV were frequently detected in patients with bronchiolitis, and hMPV was frequently associated with pneumonia. HCoV-NL63 was found to be one of the causative agents of acute respiratory wheezing in young children. No seasonal variation was found in the incidence of detection of HCoV-HKU1, HCoV-NL63 or HBoV. This 3-year study demonstrated that viral pathogens play an important role in children with ALRTIs, and more attention should be paid to these newly identified viral agents.     

WU polyomavirus infection among children in South China

This study aimed at investigating the prevalence and clinical characteristics of children with respiratory infection by WU polyomavirus (WUPyV) in Southern China. Nasopharyngeal aspirate samples were collected from 771 children with acute respiratory tract infection admitted to hospital and 82 samples from healthy subjects for routine examination at the outpatient service at the Second Affiliated Hospital of Shantou University, Medical College from July 2008 to June 2009. WUPyV was detected by the polymerase chain reaction (PCR) and DNA sequencing. All WUPyV‐positive specimens were characterized further for nine viruses causing common respiratory infections, including in?uenza A and B, respiratory syncytial virus (RSV), parainfluenza virus (PIV) 1 and 3, human metapneumovirus, human bocavirus, adenovirus, and rhinovirus by PCR or real time (RT)‐PCR. Fifteen out of 771 specimens from patients with acute respiratory tract infection, but none from healthy subjects, were positive for WUPyV and the positivity rate was 2%. Patients with WUPyV infection were between 2 and 48 months of age, and nine of the patients were male while six female. Four out of 15 patients were co‐infected with RSV, one with adenovirus or rhinovirus, respectively. Patients with WUPyV infection displayed predominantly cough, moderate fever, and wheezing, and were diagnosed with pneumonia (n = 8), bronchiolitis (n = 4), upper respiratory tract infections (n = 2) and bronchitis (n = 1). One patient developed encephalitis. Therefore, WUPyV infection can cause acute respiratory tract infection with atypical symptoms, including severe complications, in children. J. Med. Virol. 83:1440–1445, 2011. © 2011 Wiley‐Liss, Inc.     

Association of respiratory picornaviruses with acute bronchiolitis in French infants.

BACKGROUND: Human rhinoviruses and enteroviruses (Picornaviridae) are suspected to be major viral etiological causes of bronchiolitis in infants. OBJECTIVES: In the present study, we assessed the potential role of the respiratory picornaviruses as causative agents of bronchiolitis in French infants. STUDY DESIGN: From September 2001 to June 2002, we prospectively selected 192 infants < or =36 months of age and hospitalized for acute bronchiolitis. The detection of common respiratory viruses (respiratory syncytial virus, influenza virus A and B, parainfluenza virus 1, 2, 3 and adenovirus) was performed using classical immunofluorescence antigen and cell-culture detection assays on nasopharyngeal aspirates whereas the detection of human metapneumovirus (HMPV) was performed by a real-time RT-PCR assay. The presence of rhinovirus and/or enterovirus was assessed in respiratory samples by a picornavirus RT-PCR detection assay followed by a differential Southern blotting procedure. RESULTS: A potential causative virus was detected in 72.5% of the 192 study infants. RSV (30%), rhinovirus (21%), enterovirus (9%), influenza virus A (6%) and human metapneumovirus (4%) were the most frequent causative agents detected. Rhinoviruses or enteroviruses were detected as the only evidence of respiratory viral tract infection in 57 (30%) of 192 infants, whereas rhinovirus or enterovirus occurred in mixed viral infection detected in 25 (13%) of 192 study cases (30% versus 13%, p<10(-3)). CONCLUSIONS: Our data suggest that respiratory picornaviruses are one of the leading etiological causes of bronchiolitis in French infants. These findings highlight the need to implement a rapid picornavirus RT-PCR detection assay for the clinical diagnosis of respiratory infections in pediatric patients with bronchiolitis.     

长沙地区住院儿童人冠状病毒NL63的感染状况及临床特征

目的 了解HCOV-NL63在长沙地区急性下呼吸道感染住院儿童中的流行状况以及临床特征.方法 采集长沙地区2008年9月至2010年10月因急性下呼吸道感染住院儿童NPA样本1185份,用RT-PCR方法检测HCOV-NL63N基因,阳性扩增产物经测序确认,统计分析临床资料.结果 1185份NPA样本中,HCOV-NL63检出阳性10份,检出率为0.8％,其中春季检出1例、夏季检出6例、秋季检出2例、冬季检出1例;阳性检出病例患儿年龄最大2岁半,最小2个月,平均月龄12.5个月;男童7例,女童3例;临床诊断为支气管肺炎6例、毛细支气管炎3例、急性喉气管支气管炎1例;10例中有4例病情危重;4例有基础疾病;7例合并其他病毒混合感染;阳性扩增产物经测序分析与HCOV-NL63参考株同源性为97％～100％.结论 本资料HCOV-NL63阳性检出患儿年龄均在3岁以下;夏季检出率高;男童感染为主;单一基因型在长沙地区流行;HCOV-NL63可能也是我国儿童急性下呼吸道感染中的一种病原.     

Detection of viruses identified recently in children with acute wheezing

The etiologic role of recently identified respiratory viruses for acute wheezing in children is not yet clear. The purpose of this study was to investigate the prevalence of recently identified viruses, including human metapneumovirus (hMPV), human bocavirus (hBoV), human coronavirus NL63 (hCoV-NL63), and human coronavirus HKU1 (hCoV-HKU1) in children with acute wheezing. Viral etiology was identified in 231 children hospitalized with acute wheezing, aged from 1 month to 5 years. Viral antigens for common respiratory viruses were detected by IFA or multiplex PCR. RT-PCR was used to detect respiratory rhinoviruses, hCoV-NL63, hCoV-HKU1, and hMPV. PCR assays for hBoV DNA were performed using the primer sets for noncapsid protein (NP1) and nonstructural protein (NS1) genes. Viruses were found in 61.5% (142/231) of the study population and a single virus was detected in 45.5% (105/231) of the study population. Rhinovirus (33.3%), human respiratory syncytial virus (hRSV; 13.8%), and hBoV (13.8%) were the most frequently detected viruses. hMPV and hCoV-NL63 were detected in 7.8% and 1.3% of wheezing children, respectively. HCoV-HKU1 was not detected. In 16.0% of the study population, more than one virus was detected. In children with acute wheezing, rhinovirus, hRSV, and hBoV were most frequently detected. Further studies including healthy control subjects are needed to define the clinical significance of hBoV in acute wheezing.     

Etiology,seasonality, and clinical characterization of viral respiratory infections among hospitalized children in Beirut,Lebanon

Acute respiratory tract viral infections occur worldwide and are one of the major global burdens of diseases in children. The aim of this study was to determine the viral etiology of respiratory infections in hospitalized children, to understand the viral seasonality in a major Lebanese hospital, and to correlate disease severity and the presence of virus. Over a 1‐year period, nasal and throat swabs were collected from 236 pediatric patients, aged 16‐year old or less and hospitalized for acute respiratory illness. Samples collected were tested for the presence of 17 respiratory viruses using multiplex real‐time RT‐PCR. Pathogens were identified in 165 children (70%) and were frequently observed during fall and winter seasons. Co‐infection was found in 37% of positive samples. The most frequently detected pathogens were human Rhinovirus (hRV, 23%), Respiratory Syncytial Virus (RSV, 19%), human Bocavirus (hBov, 15%), human Metapneumovirus (hMPV, 10%), and human Adenovirus (hAdV, 10%). A total of 48% of children were diagnosed with bronchiolitis and 25% with pneumonia. While bronchiolitis was often caused by RSV single virus infection and hAdV/hBoV coinfection, pneumonia was significantly associated with hBoV and HP1V1 infections. No significant correlation was observed between a single viral etiology infection and a specific clinical symptom. This study provides relevant facts on the circulatory pattern of respiratory viruses in Lebanon and the importance of using PCR as a useful tool for virus detection. Early diagnosis at the initial time of hospitalization may reduce the spread of the viruses in pediatric units. J. Med. Virol. 88:1874–1881, 2016 . © 2016 Wiley Periodicals, Inc.

     

Infections and coinfections by respiratory human bocavirus during eight seasons in hospitalized children

The human bocavirus (hBoV) has been identified in respiratory infections in children in a large number of studies. Despite this, the pathogenic role of the HBoV is under discussion. The main objectives of the study were: to determine the incidence of HBoV in hospitalized children; to describe the main clinical features of the positive children; and to compare the data with those from other viral infections in the same population. A prospective study was performed between 2005 and 2013 including children up to 14‐year old with respiratory infection admitted to the Severo Ochoa Hospital (Spain). Nasopharyngeal aspirates were taken from 3,275 patients and were tested for HBoV and other 15 respiratory viruses by RT‐nested PCR. HBoV was detected in 319 patients (9.9%); 80 cases as a single pathogen, and 239 cases (75%) as coinfections with other viruses. The HBoV was the fourth most common virus detected, behind respiratory syncytial virus (39.8%), rhinovirus (30.6%), and adenovirus (15%). The most common clinical diagnosis, in cases that HBoV was detected as a single pathogen was asthma exacerbation followed by pneumonia. A seasonal distribution was shown, with higher positivity rates in December and January. Children affected by HBoV were older than children infected by other viruses. Differences in terms of clinical diagnosis were found, bronchiolitis diagnosis was lower compared with the other viruses, and HBoV was associated with diagnosis of pneumonia, with increased use of antibiotics (41.8%), and radiographic infiltrates (47%). These findings could suggest a pathogenic role of HBoV in respiratory infections in children under 14 years of age. J. Med. Virol. 88:2052–2058, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

     

Viral etiology of respiratory infections in children under 5 years old living in tropical rural areas of Senegal: The EVIRA project

Acute respiratory infection is one of the leading causes of child morbidity, especially in developing countries. Viruses are recognized as the predominant causative agents of acute respiratory infections. In Senegal, few data concerning the causes of respiratory infections are available, and those known relate mainly to classical influenza infections. Clinical and virological surveillance of acute respiratory infections was carried out in a rural community in children less than 5 years old. A standardized questionnaire was used and a nasopharyngeal swab sample was collected from each patient. These samples were tested for the detection of 20 respiratory viruses by multiplex RT‐PCR or by viral culture. A total of 82 acute respiratory episodes were included, and 48 (58.5%) were found to be positive, with a total of 55 viral detections; several samples were positive for two (n = 5) or 3 (n = 1) viruses. Ten different viruses were identified: influenza viruses A, B, and C (n = 25), human respiratory syncytial virus type A (n = 13), rhinoviruses (n = 8), human coronaviruses type 229E and NL63 (n = 6), parainfluenza viruses 3 and 4 (n = 2), and bocavirus (n = 1). These results provide evidence on the importance and the diversity of viruses as causative agents of acute respiratory infections in children living in a rural community in Senegal. The establishment of sentinel surveillance sites could help estimate the burden of acute respiratory infection in the pediatric population and should help prepare the health care systems to identify and respond to new viral respiratory emergencies. J. Med. Virol. 82:866–872, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence of human metapneumovirus in children in Argentina

Human metapneumovirus (hMPV) is a virus, which was first associated with acute lower respiratory infection in children but is detected currently in all age groups. Clinical symptoms are similar to those described for respiratory syncytial virus (RSV) infections, ranging from mild respiratory illness to severe bronchiolitis and pneumonia in children. To date, no cases of hMPV have been reported in Argentina. In this study, 440 respiratory samples obtained during the period 1998-2002 from children under 5 years old with acute respiratory infection were evaluated. Routine detection for RSV, adenovirus, influenza, and parainfluenza was undertaken by immunofluorescent assay. Of the samples negative for these viruses, only 100 were available. All these samples were tested for hMPV by RT-PCR using primers for the L gene. Eleven out of 100 (11%) respiratory samples were positive for hMPV by RT-PCR. A higher frequency of detection was observed in spring. hMPV was detected in all the years studied, except in 2001. Ten out of 11 children positive for hMPV were hospitalized. Median age was 5 months. Of seven patients, five (71%) required oxygen supplementation. The most frequent diagnosis was bronchiolitis (86%), sometimes accompanied by conjunctivitis and otitis media. The present study showed that hMPV was associated with acute lower respiratory infections in children in Buenos Aires, Argentina. This evidence strongly suggests that hMPV is a common pathogen with a wide geographical distribution, which should be included in the routine diagnosis of respiratory viruses in young children.     

Prevalence of human respiratory viruses in adults with acute respiratory tract infections in Beijing, 2005–2007

To determine the aetiological role and epidemiological profile of common respiratory viruses in adults with acute respiratory tract infections (ARTIs), a 2-year study was conducted in Beijing, China, from May 2005 to July 2007. Nose and throat swab samples from 5808 ARTI patients were analysed by PCR methods for common respiratory viruses, including influenza viruses (IFVs) A, B, and C, parainfluenza viruses (PIVs) 1–4, enteroviruses (EVs), human rhinoviruses (HRVs), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1, and adenoviruses (ADVs). Viral pathogens were detected in 34.6% of patient samples, and 1.6% of the patients tested positive for more than one virus. IFVs (19.3%) were the dominant agents detected, followed by HRVs (6.5%), PIVs (4.3%), EVs (3.2%), and HCoVs (1.1%). ADVs, RSV and HMPV were also detected (<1%). The viral detection rates differed significantly between infections of the lower and upper respiratory tracts in the sample population: PIVs, the second most commonly detected viral agents in lower acute respiratory tract infections (LRTIs), were more prevalent than in upper acute respiratory tract infections, indicating that the pathogenic role of PIVs in LRTIs should be investigated. Currently, this study is the largest-scale investigation of respiratory virus infections in China with multiple agent detection, providing baseline data for further studies of respiratory virus infections in adults with ARTIs.     

北京地区2010年10月至2011年4月急性呼吸道感染病毒流行特征分析

目的 通过对北京地区2010年10月-2011年4月急性呼吸道感染病毒病原谱构成及流行特征分析,为临床诊断、治疗和呼吸道传染病的预防控制提供病原学依据.方法 随机采集北京市五家哨点医院接诊的急性呼吸道感染病例咽拭子样本425份,利用多重RT-PCR和PCR方法进行甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、副流感病毒、...     

High incidence of the virus among respiratory pathogens in children with lower respiratory tract infection in northwestern China

Lower respiratory tract infection (LRTI) is one of the major reasons for childhood mortality that threaten the health of the public. We aimed to investigate the epidemiological pathogens and their infection analysis among children with LRTI. Sputum specimens were collected for polymerase chain reaction detection and microbiological tests to identify the viral infection and bacterial infection. The serological specimens were separated from venous blood using for Mycoplasma pneumoniae and Chlamydia pneumoniae detection. The virus was confirmed in 86.2% of the children. Human rhinovirus (38.3%), respiratory syncytial virus (32.1%), and parainfluenza virus type 3 (27.2%) were the most frequently identified pathogens. Patients with viral and bacterial coinfection showed younger age (p = 0.032), a higher proportion of wheezing rales (p = 0.032), three depressions sign (p = 0.028), and tachypnea (p = 0.038), and more likely associated with severe pneumonia (p = 0.035). Additionally, older children were more susceptible to viral-atypical bacterial coinfection (p = 0.032). Vomiting (p = 0.011) and fever (p = 0.003) were more likely to occur in children with viral-atypical bacterial coinfection. Attention should be paid to the virus infection of LRTI, as viral-bacterial coinfection and viral-atypical bacterial co-infection may have a detrimental impact on the gravity of LTRI.     

Molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in Jordan

Human respiratory syncytial virus (HRSV) is the major viral cause of acute lower respiratory tract infections in children. Few data about the molecular epidemiology of respiratory syncytial virus in developing countries, such as Jordan, are available. The frequency and severity of infections caused by HRSV were assessed in hospitalized Jordanian children <5 years of age compared with other potential etiological agents. Overall a potential pathogen was detected in 78% (254/326) of the children. HRSV was detected in 43% (140/326) of the nasopharyngeal aspirates. HRSV was found more frequently during the winter (January/February), being less frequent or negligible by spring (March/April). Analysis of 135 HRSV-positive strains using restriction fragment length polymorphism showed that 94 (70%) belonged to subgroup A, and 41 (30%) to subgroup B. There were also two cases of mixed genotypic infection. Only four of the six previously described N genotypes were detected with NP4 predominating. There were no associations between subgroup or N-genogroup and disease severity. HRSV was significantly associated with more severe acute respiratory infection and the median age of children with HRSV was lower than for those without. Next in order of frequency were adenovirus (116/312: 37%), human bocavirus (57/312: 18%), rhinovirus (36/325: 11%), Chlamydia spp. (14/312: 4.5%), human metapneumovirus (8/326: 2.5%), human coronavirus NL63 (4/325: 1.2%), and influenza A virus (2/323: 0.6%). Influenza B; parainfluenza viruses 1-4, human coronavirus HKU1 and Mycoplasma pneumoniae were not detected.