the study population comprised 402 children younger than 2  years who were hospitalized with lower respiratory tract disease at some time during march 2007july 2008 ( 248 male , mean age 11.6  months  standard deviation 6.5 ) . informed consent was obtained from parents or guardians . of the patients , 93 ( 23.2% ) this study was approved by the ethical committee of tokyo women s medical university ( # 1492 ) . on admission , the medical history and physical characteristics of the children were recorded systematically . for this study , lrti was defined as the presence of wheezing , crackling rales , dyspnea , and/or obstruction of the airways , with or without fever . pneumonia was defined as the presence of focal infiltration or consolidation in the lung by chest radiography . blood samples were obtained to measure quantities of whole white blood cells ( wbcs ) as well as neutrocytes , lymphocytes , monocytes , and eosinophils . a physician judged the bronchodilator use and administration of antibiotics for each patient according to physical findings and laboratory data . a nasopharyngeal swab was collected from each patient and then dipped into saline and stored at 80c until nucleic acid extraction . nucleic acid was extracted using a kit ( high pure viral nucleic acid kit ; roche diagnostics corp . , then pcr / reverse - transcription pcr ( rt - pcr ) was performed using previously described methods with slight modifications [ 1 , 3 , 4 , 9 , 22 , 24 ] . briefly , for the rna viruses , a superscript one - step rt - pcr system ( invitrogen corp . , carlsbad , ca , usa ) was used for reverse - transcription and first - round pcr with virus specific primers under the manufacturer s instructions . for dna viruses and shiga , japan ) was used along with the manufacturer s supplied buffer under the appropriate thermal cycle conditions for each primer . each sample was examined for rsv , rhinovirus ( rv ) , hmpv , hbov , adenovirus ( adv ) , and parainfluenzavirus ( piv ) . following amplification , pcr products were separated on the 1.5% agarose gel with tris  boric acid the pcr products were purified ; their nucleic acid sequences were determined to confirm pcr results . nucleotide sequences were processed using software ( genetyx ) and compared to nucleotide sequences of the dna database using a blast system . all data were described as mean ( m )  standard deviation ( sd ) . the study population comprised 402 children younger than 2  years who were hospitalized with lower respiratory tract disease at some time during march 2007july 2008 ( 248 male , mean age 11.6  months  standard deviation 6.5 ) . informed consent was obtained from parents or guardians . of the patients , 93 ( 23.2% ) this study was approved by the ethical committee of tokyo women s medical university ( # 1492 ) . on admission , the medical history and physical characteristics of the children were recorded systematically . for this study , lrti was defined as the presence of wheezing , crackling rales , dyspnea , and/or obstruction of the airways , with or without fever . pneumonia was defined as the presence of focal infiltration or consolidation in the lung by chest radiography . blood samples were obtained to measure quantities of whole white blood cells ( wbcs ) as well as neutrocytes , lymphocytes , monocytes , and eosinophils . a physician judged the bronchodilator use and administration of antibiotics for each patient according to physical findings and laboratory data . a nasopharyngeal swab was collected from each patient and then dipped into saline and stored at 80c until nucleic acid extraction . nucleic acid was extracted using a kit ( high pure viral nucleic acid kit ; roche diagnostics corp . , then pcr / reverse - transcription pcr ( rt - pcr ) was performed using previously described methods with slight modifications [ 1 , 3 , 4 , 9 , 22 , 24 ] . briefly , for the rna viruses , a superscript one - step rt - pcr system ( invitrogen corp . , carlsbad , ca , usa ) was used for reverse - transcription and first - round pcr with virus specific primers under the manufacturer s instructions . for dna viruses and shiga , japan ) was used along with the manufacturer s supplied buffer under the appropriate thermal cycle conditions for each primer . each sample was examined for rsv , rhinovirus ( rv ) , hmpv , hbov , adenovirus ( adv ) , and parainfluenzavirus ( piv ) . following amplification , pcr products were separated on the 1.5% agarose gel with tris  boric acid the pcr products were purified ; their nucleic acid sequences were determined to confirm pcr results . nucleotide sequences were processed using software ( genetyx ) and compared to nucleotide sequences of the dna database using a blast system . all data were described as mean ( m )  standard deviation ( sd ) . we detected at least one virus agent in 226 ( 56.2% ) of the 402 children ; 36 patients ( 9.0% ) were co - infected with different viruses . table  1viruses detected in 402 patients under 2  years of age with lower respiratory tract infectionnumberpercentpatients infected with a single pathogen22656.2%  rsv9924.6%  rv4110.2%  hmpv379.2%  hbov205.0%  piv153.7%  adv143.5%patients infected with more than one pathogen369.0%  hbov  +  piv51.2%  hbov  +  rv41.0%  hbov  +  hmpv20.5%  hbov  +  adv20.5%  hbov  +  rsv10.2%  rsv  +  rv61.5%  rsv  +  adv30.7%  piv  +  rv20.5%  hmpv  +  adv30.7%  hmpv  +  rv20.5%  hmpv  +  rsv10.2%  hmpv  +  piv10.2%  adv  +  rv10.2%  adv  +  piv10.2%  adv  +  hmpv  +  rv10.2%  rsv  +  adv  +  rv10.2%patients not infected with any studied pathogen14034.8%total402100%rsv respiratory syncytial virus , rv rhinovirus , hmpv human metapneumovirus , hbov human bocavirus , piv parainfluenza virus , adv adenovirus viruses detected in 402 patients under 2  years of age with lower respiratory tract infection rsv respiratory syncytial virus , rv rhinovirus , hmpv human metapneumovirus , hbov human bocavirus , piv parainfluenza virus , adv adenovirus during the same study period , we prospectively collected swabs from 30 age - matched children with no respiratory symptoms and analyzed them similarly . except for one patient , who was positive for rhinovirus , no virus was detected in these 30 children . in hospitalized infants and young children aged 024  months , rsv was the most commonly detected virus . of 402 patients , 111 were rsv - positive ( 27.6% ) . in 99 out of 111 patients with rsv , in 14 of 34 patients ( 41% ) , some other virus and hbov were double positive ( five with piv , four with rhinovirus , two with hmpv , two with adv , and one with rsv ) . in the other 20 patients ( 59% ) , pcr results for other viruses were negative . of the 20 patients , two showed co - infection with streptococcus pneumoniae , as diagnosed from sputum culture . we specifically examined the other 18 patients for the analysis of clinical features associated with hbov infection . may 2008 , hbov - positive patients accounted for 37% of all hospitalized children with lrtis , suggesting that hbov is a common etiologic agent causing lrtis during spring and early summer in the area of tokyo , japan ( 35n ) . b distribution of rsv - positive cases by month a distribution of human hbov - positive cases by month . b distribution of rsv - positive cases by month the median age of hbov - positive and other virus / bacteria - negative patients ( n  =  18 ) was 14  months . 2a ) ; this patient was the only one under 6  months of age who had hbov . the other two patients had chronic respiratory conditions : one was an extremely low birth weight infant developing chronic lung disease and the other with bronchial asthma . b age ( months ) of patients with detection of rsv a age ( months ) of patients with detected hbov . b age ( months ) of patients with detection of rsv of 18 patients , seven ( 39% ) were diagnosed with acute pneumonia . chest x - rays of seven patients showed reticulonodular infiltrates in one or several segments . the other 11 patients were diagnosed with acute bronchitis ( table  2 ) . table  2clinical data in children positive with hbov and in children positive with rsvhbovrsvp valuen =  18n  =  99age ( months)14.1    5.18.9    7.00.0008gender ( male % ) 7881nsdiagnosis ( pneumonia % ) 3934nsday on admission3.0    1.14.0    1.40.008oxygen saturation ( % ) 92.8    4.895.7    2.90.0004highest temperature ( c)38.9    0.838.7    1.8nswbc ( 10 cells / mm)11.4    2.911.4    4.6nsneutrophils ( % ) 62    1545    18<0.0001crp ( mg / dl)2.62    2.551.96    2.95nsdays in hospital ( days)6.6    3.26.3    1.8nscrp c - reactive protein , ns not significant , wbc white blood cell clinical data in children positive with hbov and in children positive with rsv crp c - reactive protein , ns not significant , wbc white blood cell the most common symptoms of hbov - positive patients were cough and fever , which were found in all 18 patients ( 100% ) . high fever over 38.5c was found in 13 patients ( 72% ) . hypoxia was a characteristic feature in hbov - positive patients ( table  2 ) . on admission , the mean oxygen saturation in hbov - positive patients was 92.8    4.8% . the mean period from onset of illness to admission was 3.0    1.1  days . for 17 of 18 patients , the main reason for admission was hypoxia and/or respiratory distress . the clinical course was good except for that of one infant , who had extremely low birth weight and had chronic lung disease . we compared clinical data of the 18 patients with the data of hospitalized patients who were positive for rsv and negative for other viruses in the same study period ( n  =  99 , table  2 ) . one patient had hbov at less than 6  months of age , although 44 patients with rsv ( 44% ) were less than 6  months of age ( fig . the period from disease onset to admission was significantly shorter among hbov - positive patients than among rsv - positive patients ( p  =  0.008 , table  2 ) . furthermore , the mean oxygen saturation in hbov - positive patients on admission was significantly lower than that in rsv - positive patients ( p  =  0.0004 , table  2 ) . however , the percentage of neutrophils in wbcs were significantly higher in hbov - positive patients than in rsv - positive patients ( p  <  0.0001 , table  2 ) . the c - reactive protein in hbov - positive patients was similar to that of rsv - positive patients ( table  2 ) . we detected at least one virus agent in 226 ( 56.2% ) of the 402 children ; 36 patients ( 9.0% ) were co - infected with different viruses . table  1viruses detected in 402 patients under 2  years of age with lower respiratory tract infectionnumberpercentpatients infected with a single pathogen22656.2%  rsv9924.6%  rv4110.2%  hmpv379.2%  hbov205.0%  piv153.7%  adv143.5%patients infected with more than one pathogen369.0%  hbov  +  piv51.2%  hbov  +  rv41.0%  hbov  +  hmpv20.5%  hbov  +  adv20.5%  hbov  +  rsv10.2%  rsv  +  rv61.5%  rsv  +  adv30.7%  piv  +  rv20.5%  hmpv  +  adv30.7%  hmpv  +  rv20.5%  hmpv  +  rsv10.2%  hmpv  +  piv10.2%  adv  +  rv10.2%  adv  +  piv10.2%  adv  +  hmpv  +  rv10.2%  rsv  +  adv  +  rv10.2%patients not infected with any studied pathogen14034.8%total402100%rsv respiratory syncytial virus , rv rhinovirus , hmpv human metapneumovirus , hbov human bocavirus , piv parainfluenza virus , adv adenovirus viruses detected in 402 patients under 2  years of age with lower respiratory tract infection rsv respiratory syncytial virus , rv rhinovirus , hmpv human metapneumovirus , hbov human bocavirus , piv parainfluenza virus , adv adenovirus during the same study period , we prospectively collected swabs from 30 age - matched children with no respiratory symptoms and analyzed them similarly . except for one patient , who was positive for rhinovirus , no virus was detected in these 30 children . in hospitalized infants and young children aged 024  months , rsv was the most commonly detected virus . of 402 patients , 111 were rsv - positive ( 27.6% ) . in 99 out of 111 patients with rsv , in 14 of 34 patients ( 41% ) , some other virus and hbov were double positive ( five with piv , four with rhinovirus , two with hmpv , two with adv , and one with rsv ) . in the other 20 patients ( 59% ) , pcr results for other viruses were negative . of the 20 patients , two showed co - infection with streptococcus pneumoniae , as diagnosed from sputum culture . we specifically examined the other 18 patients for the analysis of clinical features associated with hbov infection . may 2008 , hbov - positive patients accounted for 37% of all hospitalized children with lrtis , suggesting that hbov is a common etiologic agent causing lrtis during spring and early summer in the area of tokyo , japan ( 35n ) . b distribution of rsv - positive cases by month a distribution of human hbov - positive cases by month . the median age of hbov - positive and other virus / bacteria - negative patients ( 2a ) ; this patient was the only one under 6  months of age who had hbov . the other two patients had chronic respiratory conditions : one was an extremely low birth weight infant developing chronic lung disease and the other with bronchial asthma . b age ( months ) of patients with detection of rsv a age ( months ) of patients with detected hbov . b age ( months ) of patients with detection of rsv of 18 patients , seven ( 39% ) were diagnosed with acute pneumonia . chest x - rays of seven patients showed reticulonodular infiltrates in one or several segments . the other 11 patients were diagnosed with acute bronchitis ( table  2 ) . table  2clinical data in children positive with hbov and in children positive with rsvhbovrsvp valuen  =  18n  =  99age ( months)14.1    5.18.9    7.00.0008gender ( male % ) 7881nsdiagnosis ( pneumonia % ) 3934nsday on admission3.0    1.14.0    1.40.008oxygen saturation ( % ) 92.8    4.895.7    2.90.0004highest temperature ( c)38.9    0.838.7    1.8nswbc ( 10 cells / mm)11.4    2.911.4    4.6nsneutrophils ( % ) 62    1545    18<0.0001crp ( mg / dl)2.62 2.551.96    2.95nsdays in hospital ( days)6.6    3.26.3    1.8nscrp c - reactive protein , ns not significant , wbc white blood cell clinical data in children positive with hbov and in children positive with rsv crp c - reactive protein , ns not significant , wbc white blood cell the most common symptoms of hbov - positive patients were cough and fever , which were found in all 18 patients ( 100% ) . hypoxia was a characteristic feature in hbov - positive patients ( table  2 ) . on admission , the mean oxygen saturation in hbov - positive patients was 92.8    4.8% . the mean period from onset of illness to admission was 3.0    1.1  days . for 17 of 18 patients , the main reason for admission was hypoxia and/or respiratory distress . the clinical course was good except for that of one infant , who had extremely low birth weight and had chronic lung disease . we compared clinical data of the 18 patients with the data of hospitalized patients who were positive for rsv and negative for other viruses in the same study period ( n  =  99 , table  2 ) . however , rsv was identified all year round ; its epidemic peak was november  december ( fig . one patient had hbov at less than 6  months of age , although 44 patients with rsv ( 44% ) were less than 6  months of age ( fig . the period from disease onset to admission was significantly shorter among hbov - positive patients than among rsv - positive patients ( p  =  0.008 , table  2 ) . furthermore , the mean oxygen saturation in hbov - positive patients on admission was significantly lower than that in rsv - positive patients ( p  =  0.0004 , table  2 ) . however , the percentage of neutrophils in wbcs were significantly higher in hbov - positive patients than in rsv - positive patients ( p  <  0.0001 , table  2 ) . the c - reactive protein in hbov - positive patients was similar to that of rsv - positive patients ( table  2 ) . this prospective study reveals several characteristics of hbov - positive lrti in young children : seasonality , age distribution , hypoxia , neutrophilia , and findings in chest radiography . comparison of clinical features to those in patients with rsv infection , which is the major etiologic agent in lrti of young children , showed that major clinical manifestations such as wheezing and respiratory distress were similar in both groups of patients . however , several physical characteristics of hbov - positive patients differed significantly from those of rsv - positive patients . to describe clinical manifestations of children with lrtis with hbov clearly , we selected hbov - positive and other virus / bacteria - negative patients . regarding the hbov seasonality , its peak was similar to that identified in a previous study in northern japan . in the study , the seasonal distribution is november  june ; the peak occurs in march  april , which resembles the circumstances in japan . in hong kong and thailand , the peak seasons were in fall and winter , respectively [ 8 , 14 ] . specific examination of extremely young patients revealed a clear difference in the age distribution of hbov - positive patients from that of rsv . in rsv - positive patients , in contrast , the minimum age of hbov - positive patients was 5  months . endo et al . measured the levels of the antibody in human serum samples aged from 0  months to 41  years and reported that the seropositive rate was lowest in the age group of 68  months . these data suggest that infants younger than 6  months might be protected from hbov infection . the main clinical features in hbov - positive patients are respiratory distress and hypoxia , which are frequently observed in lrti patients with rsv . in our study , oxygen saturation on admission shows that hypoxia was more severe in hbov - positive patients than in rsv - positive patients . moreover , the short duration from disease onset to admission shows that respiratory distress and hypoxia progress rapidly in hbov - positive patients . recently reported a severe case with hbov associated with plastic bronchitis , which necessitated mechanical ventilation . many previous reports have not described severe cases of hbov infection ; however , the sample size is small and the studies were retrospective . in our study , . a larger number of cases with hbov - positive lrti must be accumulated to discuss the disease severity . one reason is that the rsv infection is frequently found in infants who are younger than 3  months . in contrast , the minimum age of hbov - positive patient is 5  months in our study . laboratory data show that the rate of neutrophils in wbcs is significantly higher in hbov - positive patients , which is a unique characteristic of hbov - positive lrti . it is particularly interesting that the rate of neutrophils in hmpv - positive patients is similar to that in rsv - positive patients ( data not shown ) . acute hypoxia induces a differential response in peripheral blood neutrophils and lymphocytes , resulting in neutrophilia and lymphopenia . recently , a high co - infection rate is under discussion in hbov - positive respiratory infection [ 17 , 18 ] . previous reports have presented the possibility that hbov detection in respiratory tract samples reflects asymptomatic persistence or prolonged viral shedding . although results of the present study also show a high co - infection rate , clinical characteristics in hbov - single positive patients strongly suggest the pathogenic potential of hbov in young children with lrtis . quantification of viral loads might provide useful information , especially information that would be useful to analyze co - infection cases more clearly . in conclusion , the results of our study support that hbov is an important causative agent in young children hospitalized with lrti in japan . in young children , more cases should be examined to confirm clinical manifestations and prognoses of hbov - associated lrtis .
background and objectiveclinical characteristics of human bocavirus ( hbov ) infection have been studied worldwide , but their importance of those characteristics remains unknown . 
 we investigated distinctive clinical features of hbov - positive children with lower respiratory tract infection ( lrti).methods and resultsduring april 2007july 2009 , for 402 hospitalized children younger than 2  years with lrti , we prospectively examined virus genomes in nasopharyngeal swabs for hbov , respiratory syncytial virus ( rsv ) , rhinovirus , metapneumovirus , parainfluenzavirus , and adenovirus . 
 the hbov genomes were identified in 34 patients ( 8.5% ) . 
 clinical and laboratory data of hbov - positive and other virus / bacteria - negative patients ( n  =  18 ) were analyzed and compared with data of rsv - single positive patients ( n  =  99 ) . 
 the seasonal distribution of hbov exhibits a concentration of cases during march  september , with most rsv cases occurring during winter in japan . 
 the minimum age of hbov - positive patients was 5  months , although 44 patients ( 44% ) with rsv were younger than 6  months . 
 the main clinical features were respiratory distress and hypoxia . 
 hypoxia advances within 3  days after onset . 
 the mean oxygen saturation on arrival was 92.8% , which was significantly lower than that in patients with rsv ( p  <  0.001 ) . 
 white blood cell counts were similar among groups . 
 however , the percentage of neutrophils in white blood cells were significantly higher in hbov - positive patients ( 62 vs. 45% , p  <  0.001 ) . 
 their prognoses were good . 
 their hospital stays were 6.6  days.conclusionshbov-single positive patients show several clinical characteristics , such as seasonality , age , hypoxia , and neutrophilia , which differ from those with rsv infection .