the influenza a h1n1 ( 2009 ) virus affected thousands of children in catalonia ( spain ) in fall 2009 ( 10001200 infections per 100  000 inhabitants aged < 15year old between weeks 43 and 46 of 2009 ) . 
 
the infection typically caused mild respiratory symptoms , but other less usual extrapulmonary manifestations and a severe clinical course were observed even in previously healthy patients . 

, 
  the attack rate of influenza a h1n1 ( 2009 ) during the first postpandemic season has been much lower , nevertheless the virus has continued to cause serious illness in children
. 

  viral load may provide important information about the interaction between the infective agent and the host .
the information about the possible correlation between viral load , epidemiological characteristics and the severity of disease in pediatric patients is scarce .
the aim of this study was to describe influenza a h1n1 ( 2009 ) viral load at diagnosis ( vlad ) and its relation to epidemiological , clinical characteristics and laboratory parameters in a series of hospitalized pediatric patients .
we also wanted to assess viral load at diagnosis as a biomarker of disease severity .
the study was conducted in a 345bed tertiary care pediatric hospital with a 18bed pediatric intensive care unit , from weeks 3051 of 2009 . during the study period ,
patients with respiratory symptoms who required hospital admission were tested for influenza a h1n1 ( 2009 ) .
patients < 18  years old with influenza a h1n1 ( 2009 ) infection and hospitalized with respiratory symptoms were consecutively included .
severe cases were defined as those who required invasive or noninvasive mechanical ventilation ( mv ) because of respiratory failure or severe respiratory distress .
total nucleic acids were extracted from nasopharyngeal aspirate with the roche magna pure compact system ( roche molecular diagnostics , mannheim , germany ) according to manufacturer s protocol .
the influenza a h1n1 ( 2009 ) infection was confirmed by detection of matrix protein 2 ( m2 ) and pandemic hemagglutinin h1 genes by using a realtime reverse transcriptase polymerase chain reaction ( realtime rtpcr ) ( realtime ready inf a / h1n1 detection set ; roche molecular diagnostics ) .
this test has a detection limit of 10  copies per pcr reaction . for the quantitative assay ,
10 fold dilutions equivalent to 1010    10 copies per reaction of a plasmid positive control for m2 gene positive control supplied by the manufacturer were prepared to generate a calibration curve .
viral load was retrospectively determined through cyclethreshold ( c 
 t ) values of m2 gene corrected according to the c 
 t value of internal control ( human myostatine gene ) .
nasopharyngeal aspirates with an internal control c 
 t  >  27 were considered as poor quality samples for viral load determination 
 .
the reactions were performed and analyzed by using the lightcycler 480 system ( roche molecular diagnostics ) .
descriptive statistics of noncontinuous variables were reported in terms of absolute frequencies and percentages , and data comparisons were performed using pearson chisquare test or fisher exact test when the expected count in any category was < 5 .
continuous nonnormally distributed variables were described in terms of median value with interquartile range ( iqr , 25th percentile75th percentile ) and compared using mann 
the receiveroperator characteristic ( roc ) curve analysis was performed with vlad distinguishing between patients who required mv and those who did not required mv . 

spss 
  software v 19.0 for windows 
  was used to perform the statistical tests . for the analysis ,
total nucleic acids were extracted from nasopharyngeal aspirate with the roche magna pure compact system ( roche molecular diagnostics , mannheim , germany ) according to manufacturer s protocol .
the influenza a h1n1 ( 2009 ) infection was confirmed by detection of matrix protein 2 ( m2 ) and pandemic hemagglutinin h1 genes by using a realtime reverse transcriptase polymerase chain reaction ( realtime rtpcr ) ( realtime ready inf a / h1n1 detection set ; roche molecular diagnostics ) .
this test has a detection limit of 10  copies per pcr reaction . for the quantitative assay ,
10 fold dilutions equivalent to 1010    10 copies per reaction of a plasmid positive control for m2 gene positive control supplied by the manufacturer were prepared to generate a calibration curve .
viral load was retrospectively determined through cyclethreshold ( c 
 t ) values of m2 gene corrected according to the c 
 t value of internal control ( human myostatine gene ) .
nasopharyngeal aspirates with an internal control c 
 t  >  27 were considered as poor quality samples for viral load determination 
 .
the reactions were performed and analyzed by using the lightcycler 480 system ( roche molecular diagnostics ) .
descriptive statistics of noncontinuous variables were reported in terms of absolute frequencies and percentages , and data comparisons were performed using pearson chisquare test or fisher exact test when the expected count in any category was < 5 .
continuous nonnormally distributed variables were described in terms of median value with interquartile range ( iqr , 25th percentile75th percentile ) and compared using mann 
the receiveroperator characteristic ( roc ) curve analysis was performed with vlad distinguishing between patients who required mv and those who did not required mv . 

spss 
  software v 19.0 for windows 
  was used to perform the statistical tests . for the analysis ,
during the study period , 93 children were admitted with respiratory distress and/or hypoxemia and influenza a h1n1 ( 2009 ) infection .
twentythree patients were excluded because either their nasopharyngeal aspirate sample had an internal control c 
 t  >  27 ( 11/93 ) or they were diagnosed in other institutions ( 12/93 ) and were not retested .
viral load was determined within a median of 3  days after onset of respiratory symptoms ( iqr : 15 ) .
we established two different groups regarding to vlad and duration of illness at the moment of sampling : those who had been with symptoms for four or less days and those who had symptoms for five or more days ( median , 60 log10  copies / ml ( iqr : 4668 ) versus 47 ( 3957 ) ; p  =  002 ) ( figure  1 ) .
the viral load was inversely correlated to the days after the onset of fever ( spearman rho  =  036 ; p  <  001 ) . 
regarding to the association with laboratory parameters , vlad in patients within first 4  days of symptoms was inversely correlated with the number of lymphocytes at diagnosis ( spearman rho  =  047 ; p  <  001 ) , but there was no correlation in those diagnosed with 5  days of symptoms . viral load was not correlated with values of c reactive protein .
neither was it with absolute neutrophil count or with hemoglobin ( data not shown ) . 
influenza a h1n1 ( 2009 ) viral load at diagnosis ( vlad ) according to demographic characteristics and underlying comorbidity * median ( interquartile range ) . *
* male versus female . * * * < 5year old versus 5year old . 
 in comparison with previously healthy patients .
twenty patients required mv because of respiratory failure or severe respiratory distress during the hospitalization .
the roc curve analysis was used to analyze the accuracy of vlad as a biomarker of severity ( need of mv ) . in patients within the first 4  days of symptoms ,
the area under the roc curve was 055 ( 95% confidence interval , 039069 ; p 
the area under the roc curve of vlad was 073 ( 95% confidence interval , 051088 ; p 
the cutoff value of 45 log10  copies / ml corresponded with the highest average of sensitivity ( 100% , 95% ci : 631100 ) and specificity ( 65% , 95% ci : 383858 ) .
the negative predictive value of a lower value of viral load for distinguishing patients who required mv was 100% ( 95% ci : 715100 ) . having <
45 log10  copies / ml with 5  days of clinical symptoms was associated with a lower risk of requiring mv [ relative risk ( rr )  =  013 ( 95% ci : 002092 ) ] .
patients who were with symptoms for 5  days and with a vlad 45 log10  copies / ml had an increased risk of requiring mv in comparison with those that exceeded that viral load and were still within the first 4  days of symptoms [ rr  =  208 ( 95% ci : 107405 ) ] .
the number of deceased patients was too low ( n  =  4 ) to draw any conclusion regarding differences in vlad .
all of them had a vlad  >  45 log10  copies / ml .
most of the patients ( 66/70 , 94% ) received oseltamivir during hospitalization immediately after collecting the sample for viral load determination .
children had been widely described as a population with an increased risk of severe disease by influenza a h1n1 ( 2009 ) . the immunopathology may play an important role in the severely affected children 
  apart from the virusmediated component of disease , which is the focus of this study .
the literature published about these two components of the novel influenza disease in hospitalized children is scarce . viral load at diagnosis may be a biomarker of severity , as described previously for other respiratory virus , including h5n1 influenza virus 
 , 

, 
  vlad can be easily extrapolated with the same realtime rtpcr used for the diagnosis of infection without almost adding any extra economic cost . in our study ,
vlad within the first days of symptoms was not a good biomarker of severity . nonetheless in patients with 5 or more days of symptoms ,
vlad was an accurate parameter distinguishing the patients who required mv from those who did not required it .
it is known that the lag time between the onset of symptoms and the initiation of antiviral treatment determines a different clinical response in influenza a h1n1 ( 2009 ) infections . 

, 
  in patients within the first days of symptoms , early initiation of oseltamivir could have protected them from developing a more severe respiratory disease , and probably this may be one of the reasons for not finding the vlad values as an accurate parameter in distinguishing the most severe cases in that group . on the other hand , the treatment has proved to be effective in decreasing viral load , 
  suggesting that those patients with high viral loads after several days of symptoms could have benefited from an earlier treatment . in this series , as reported previously for seasonal and pandemic influenza , 
 , 
 , 
  vlad was at high level in those patients in whom sample was collected during the first days of symptoms . higher influenza a h1n1 ( 2009 ) viral loads had been observed in patients with fever and in those with pneumonia 
 , 
  in comparison with patients without systemic symptoms .
. 

  observed a lower clearance of viral load in patients younger than 13year old , but they did not find in them a higher rate of severe disease , defined in that study as having chest xray opacities or meningoencephalitis . in our series , to have a 
high vlad with 5  days of symptoms conferred a twofold increase in the risk of mv .
this result could be in relation with the observed lower viral load clearances in patients with more severe disease in other series . 
 , 
 , 

  the main limitation of the study is the small sample size .
detection of oseltamivirresistant strains was not systematically performed , although resistant strains were a minority during the pandemic period . 
 
it would also be interesting to determine the clinical utility of serial analysis of viral load for each individual case , but it may be difficult to be introduced as a regular clinical practice in pediatrics . based on our observations ,
we conclude that vlad in patients with several days of clinical symptoms (   5  days in our study ) was associated with the severity of influenza a h1n1 ( 2009 ) disease .
further studies in different settings are needed to identify the weighted role of the direct viral damage and the immunopathology in pediatric patients with influenza a h1n1 ( 2009 ) disease .
 
 please cite this paper as : launes et  al . 
 ( 2012 ) viral load at diagnosis and influenza a h1n1 ( 2009 ) disease severity in children . 
 influenza and other respiratory viruses 6(601 ) , e89e92.to assess viral load at diagnosis ( vlad ) as a biomarker of novel influenza disease severity , epidemiologic and clinical data of admitted patients < 18  years old with influenza a h1n1 ( 2009 ) infection and respiratory symptoms were prospectively collected in a single pediatric tertiary hospital , from weeks 3051 of 2009 . 
 seventy patients were included . 
 vlad in children who had symptoms for 5  days was an accurate parameter distinguishing the patients who required mechanical ventilation ( mv ) from those who did not required it ( area under the roc curve : 073 ; p  =  003 ) . having < 45  log10  copies / ml with 5  days of symptoms was associated with a lower risk of requiring mv . 
Introduction
Methods
Diagnosis and quantification of viral load
Statistical analysis
Results
Discussion
Competing Interests